Humanized anti-CD40 antibodies and uses thereof

Abstract

To provide therapies that specifically target the inununological pathways involved in transplant rejection reaction and autoimmune disorders.SOLUTION: The present disclosure relates to anti-CD40 antibodies, such as humanized anti-CD-40 antibodies, that may be used in various therapeutic, prophylactic and diagnostic methods. The antibodies generally block the ability of CD40 to bind CD154 and do so without activating the cell expressing CD40 (e.g., a B cell). The present antibodies or fragments thereof may be used to reduce complications associated with organ or tissue transplantation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 214,411, filed September 4, 2015. This application claims priority from US Provisional Application No. 60 / 019,599, filed on May 1, 2006, the disclosure of which is incorporated herein by reference in its entirety. will be incorporated into

[0002] Sequence Listing This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference. The ASCII copy of the foregoing is incorporated herein by reference in its entirety. It was created on the 1st of the month and is named 11212-005211-WO0_SL.tx t and is 33,948 bytes in size.

[0003] The present invention provides humanized anti-CD40 antibodies and methods for treating, for example, transplant rejection, including the use of humanized anti-CD40 antibodies. or to treat transplant rejection, to induce immunosuppression, or to treat autoimmune disorders. The present invention relates to the use of such antibodies for detecting leukemia cells. [Background technology]

[0004] Suppression of the immune system, particularly the humoral immune system, is useful in organ transplantation and the treatment of autoimmune disorders. Organ transplantation is the preferred treatment for many forms of life-threatening disease involving organ damage. However, the transplanted cells or tissues have been shown to be ineffective in the recipient organism. If an unwanted immune response occurs against the tissue, transplant rejection can occur. Rejection can be minimized by tissue type matching, but even with matched tissue, Therefore, virtually all tissue transplants are now performed using a donor-specific donor-specific donor system. Immunosuppressive therapy is used in cases of transplantation.

[0005] The improvement in clinical transplant outcomes is primarily due to increasingly powerful methods for inhibiting rejection responses. Short-term outcomes have improved with the development of nonspecific immunosuppressants. However, long-term outcomes remain unsatisfactory. Lifelong immunosuppressant medications may be required for these conditions, and the use of these medications can have cardiovascular and Dramatically increases the risk of disease, infection and malignancy.

[0006] One potential target for reducing transplant rejection is the CD40 / CD154 interaction. CD40 is expressed primarily on B lymphocytes and other antigen-presenting cells (APCs), such as dendritic cells. CD154 is expressed primarily on the surface of T cells. The interaction between these two proteins regulates cytokine expression and cell surface markers. , which are associated with B cell activation, inducing the expression of, for example, CD23, CD80, and CD86. Ru. Kehry MR,CD40-mediated signaling in B cells.Balancing cell survival,growth,a nd death.J.Immunol.1996;156:2345-2348. anti-C Blocking this interaction with the D154 antibody resulted in improved survival of transplanted non-human primates. Tissue rejection has been shown to be reduced or eliminated. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kehry MR,CD40-mediated signaling in B cells.Balancing cell survival,growth, and death.J.Immunol.1996;156:2345-2348 Summary of the Invention [Problem to be solved by the invention]

[0008] For any type of immunosuppression (e.g., in transplantation procedures), a balance between efficacy and toxicity must be struck. are the main factors for its clinical acceptability. There is a need for therapeutic agents that specifically target immunological pathways involved in immune disorders. There are. [Means for solving the problem]

[0009] The present disclosure provides a humanized anti-CD40 antibody or antigen-binding portion thereof comprising a heavy chain variable region. and the heavy chain variable region has approximately the same amino acid sequence as shown in SEQ ID NOs: 13, 14 and 15, respectively. Three CDRs, CDR1, CDR2, and CDR3, which have 80% to about 100% identical amino acid sequences A humanized anti-CD40 antibody or antigen-binding portion thereof is provided that comprises a CDR3.

[0010] The present disclosure also provides a humanized anti-CD40 antibody or antigen-binding portion thereof comprising a light chain variable region. wherein the light chain variable region has the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively. Three CDRs, CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR110, CDR111, CDR12, CDR13, CDR14, CDR15, CDR16 and a humanized anti-CD40 antibody or antigen-binding portion thereof, which comprises CDR2 and CDR3. .

[0011] The present disclosure also provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or The antigen-binding portion, wherein the heavy chain variable region is as set forth in SEQ ID NOs: 13, 14 and 15, respectively. Three complementarity-determining regions with amino acid sequences that are approximately 80% to 100% identical to the amino acid sequence of the light chain variable region comprises CDR1, CDR2 and CDR3, Approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively and a CDR1, a CDR2, and a CDR3 having the amino acid sequence Also encompassed are humanized anti-CD40 antibodies or antigen-binding portions thereof.

[0012] The present disclosure provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or an antigen thereof. A binding moiety, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 11, 19, 20, 21, 24, 25 and an amino acid sequence that is about 80% to about 100% identical to any one of the amino acid sequences shown in 26. The present invention provides a humanized anti-CD40 antibody or antigen-binding portion thereof, comprising:

[0013] The present disclosure provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or an antigen thereof. A binding moiety, wherein the light chain variable region is selected from the group consisting of SEQ ID NOs: 12, 22, 23, 27, 28 and 29. 9. The present invention provides a humanized anti-CD40 antibody or antigen-binding portion thereof, which is

[0014] The present disclosure provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or an antigen thereof. A binding moiety, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 11, 19, 20, 21, 24, 25 and an amino acid sequence that is about 80% to about 100% identical to any one of the amino acid sequences shown in 26. The light chain variable region comprises SEQ ID NOs: 12, 22, 23, 27, 28 and 29. 9. The present invention provides a humanized anti-CD40 antibody or antigen-binding portion thereof, which is

[0015] The present disclosure provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or an antigen thereof. A binding moiety, wherein the heavy chain variable region has the amino acid sequences shown in SEQ ID NOs: 19, 20 and 21. and the light chain comprises an amino acid sequence that is about 80% to about 100% identical to any one of the light chains. The variable region has about 80% to about 10% identical amino acid sequence to either of the amino acid sequences shown in SEQ ID NOs: 22 and 23. a humanized anti-CD40 antibody or its antigen-binding portion that contains an amino acid sequence that is 100% identical to that of the humanized anti-CD40 antibody or its antigen-binding portion; provide.

[0016] The present disclosure provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or an antigen thereof. A binding moiety, wherein the heavy chain variable region has the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26. and the light chain comprises an amino acid sequence that is about 80% to about 100% identical to any one of the light chains. The variable region has about 80 amino acid sequences identical to any one of the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29. Humanized anti-CD40 antibodies or their antigens containing amino acid sequences that are 100% to about 100% identical Provides a binding moiety.

[0017] The present disclosure provides a humanized anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, or an antigen thereof. a binding portion, wherein the heavy chain variable region has an amino acid sequence identical to that of SEQ ID NO: 21 at a ratio of about 80% to 100%; and a light chain variable region comprising an amino acid sequence approximately 100% identical to that of SEQ ID NO: 23. A humanized antibody containing an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence shown in CD40 antibodies or antigen-binding portions thereof are provided.

[0018] The dissociation constant (K D ) is approximately 1 × 10 -9 Less than M or about 1 x 1 0 -8 It can be less than M.

[0019] The antibodies or antigen-binding portions thereof of the present invention are: (a) intact immunoglobulin molecules; (b) s (c) a cFv; (c) a Fab fragment; (d) a F(ab')2; and / or (e) a disulfide bond It can be Fv.

[0020] The antibodies of the invention, or antigen-binding portions thereof, comprise: a) the constant domain of IgG; and (b) the constant domain of IgA The antibody may comprise at least one constant domain selected from the constant domains of:

[0021] The antibodies of the present invention, or antigen-binding portions thereof, comprise at least one human constant domain. could be.

[0022] The antibodies of the present invention, or antigen-binding portions thereof, are capable of binding to the CD40 extracellular domain. .

[0023] The CD40 can be human or rhesus CD40.

[0024] The antibodies of the invention, or antigen-binding portions thereof, bind to B lymphocytes mediated by CD154-expressing Jurkat cells. It is capable of blocking lymphocyte activation in vitro.

[0025] The antibodies or antigen-binding portions thereof of the present invention bind to CD23, CD80, or CD86 on B lymphocytes. The present invention is capable of inhibiting the expression of the

[0026] Also, the present invention relates to a method for producing a composition comprising an antibody or an antigen-binding portion thereof of the present invention and at least one pharmaceutically acceptable carrier. Compositions comprising such carriers are also encompassed by the present disclosure.

[0027] In accordance with the present disclosure, polynucleotides encoding the antibodies of the invention or antigen-binding portions thereof are provided. The present disclosure provides vectors comprising the polynucleotides of the present invention, and The present invention provides a cell comprising:

[0028] The present disclosure provides an isolated polypeptide comprising an antibody of the invention or an antigen-binding portion thereof. .

[0029] Also encompassed by the present disclosure are methods for producing the antibodies, or antigen-binding portions thereof, of the present invention. the steps of: (a) culturing the cells of the present invention in a culture medium with an antibody or antigen-binding portion thereof of the present invention; and culturing the cells under conditions in which the polynucleotide encoding the antibody or producing at least one polypeptide comprising the antigen-binding portion; and 2.) recovering the polypeptide from the cells or culture medium.

[0030] The present disclosure also provides a method for treating a cancer by administering to a subject an effective amount of an antibody of the invention, or an antigen-binding portion thereof. The present invention provides a method for suppressing the immune system of a subject, comprising the steps of:

[0031] The present disclosure provides a method for treating or preventing transplant rejection, or preventing transplant rejection from occurring. 1. A method for increasing the duration of a steroid hormone in a subject in need thereof, comprising administering to said subject A method is provided that includes administering to the body an effective amount of an antibody of the invention, or an antigen-binding portion thereof.

[0032] The present disclosure provides for the treatment or prophylactic treatment of graft-versus-host disease in a subject in need thereof. administering to said subject an effective amount of an antibody of the invention or an antigen-binding portion thereof, The present invention provides a method comprising the steps of:

[0033] The present disclosure provides for the treatment or prophylactic treatment of autoimmune disorders in a subject in need thereof. administering to said subject an effective amount of an antibody of the invention or an antigen-binding portion thereof, The present invention provides a method comprising the steps of:

[0034] The subject may be a subject who has undergone an organ transplant and / or tissue transplant, or and / or subjects in need of tissue transplantation. Organs include heart, kidney, lung, liver, pancreas The tissue may be bone, tendon, cornea, skin, heart valves, veins or can be bone marrow.

[0035] The subject can be a human or mammal.

[0036] The administration may begin before transplantation. The administration may continue for at least one month after transplantation. The administration may be continued for at least six months after transplantation of the graft.

[0037] An autoimmune disorder can be one that is associated with the presence of autoantibodies or It can be caused by existence.

[0038] Autoimmune disorders include systemic lupus erythematosus (SLE), CREST syndrome (calcification syndrome), syndrome, Raynaud's syndrome, esophageal motility disorders, sclerodactyly and capillaries ectasia), opsoclonus, inflammatory myopathies (e.g., polymyositis, dermatomyositis, and cerebrospinal fluid purpura) Myositis), systemic sclerosis, primary biliary cirrhosis, celiac disease (e.g., gluten intolerance) enteropathy), dermatitis herpetiformis, Miller-Fisher syndrome, acute motor axonal neuropathies Qi (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, anti Phospholipid syndrome, Wegener's granulomatosis, microscopic polyangiitis is), Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmune hepatitis, sclerosing myositis (scleromyositis), myasthenia gravis, Lambert-Eaton myasthenic syndrome , Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, stiff-person syndrome, limbic encephalitis, Isaacs syndrome, Sydenham's chorea, pediatric autoimmune streptococcal neuropsychiatric disorder The cause may be pan-and-associated decompensated asthma (PANDAS), encephalitis, type 1 diabetes mellitus and / or neuromyelitis optica.

[0039] Autoimmune disorders include pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease, psoriatic arthritis, and psoriasis. Lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and new The cause may be fetal lupus erythematosus), multiple sclerosis, and / or reactive arthritis.

[0040] Autoimmune disorders include polymyositis, dermatomyositis, polyendocrine deficiency, Schmidt syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, Lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating disease, subacute cutaneous erythematous lupus Acne, hypoparathyroidism, Dressler syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenia Hypoplastic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia areata, pemphigoid Acne, scleroderma, systemic progressive sclerosis, adult-onset diabetes mellitus (e.g. type II diabetes), male Sexual and female autoimmune infertility, ankylosing spondylitis (spondolytis), ulcerative colitis Colitis, Crohn's disease, mixed connective tissue disease, polyarteritis nodosa, systemic necrosis factor syndrome Deadly vasculitis, juvenile-onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis , Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent Abortion, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-open heart syndrome, Cushing's syndrome, Autoimmune chronic active hepatitis, bird enthusiast's disease, allergic disease, allergic encephalomyelitis, toxicity Epidermal necrolysis, alopecia, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis , interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reactions, leprosy, malaria, Lee Osteoarthritis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis Hemophiliasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's disease s) syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Kaplan's syndrome, Kawasaki disease, Fever, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum elevatum et diutinum), erythroblastosis fetalis, eosinophilic fasciitis (fac iitis), Shulman syndrome, Felty syndrome, filariasis, cyclitis, chronic trichomeitis Cycloiditis, metachronous cyclitis, Fuchs' cyclitis, IgA nephropathy, Henoch-Schönlein purple Plaque disease, graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus infection , cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Hodgkin's and non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia , Waldenström's macroglobulinemia, Epstein-Barr virus infection, Otafuku The cause may be the common cold, Evans syndrome, and / or autoimmune dysgonadism.

[0041] The administration may be parenteral, intravenous, subcutaneous, intramuscular, transdermal, oral, topical, intrathecal, or local. obtain.

[0042] The methods of the invention further comprise administering an antibody or antigen-binding portion thereof of the invention to a subject immunized within six months of administration. This may involve the administration of a suppressant drug.

[0043] Immunosuppressants include calcineurin inhibitors, tacrolimus, mTor inhibitors, and fingolimod. Modo, myriocin, alemtuzumab, rituximab, anti-CD4 monoclonal antibody, anti LFA1 monoclonal antibody, anti-LFA3 monoclonal antibody, anti-CD45 antibody, anti-CD 19 antibodies, monabatacept, belatacept, indolyl-ASC; azathioprine, phosphorus Thymocyte immunoglobulin and antithymocyte globulin [horse], mycophenolate mofetil, Mycophenolate sodium, daclizumab, basiliximab, cyclophosphamide, Rednisone, prednisolone, leflunomide, FK778, FK779, 15-deoxy Spergualin, busulfan, fludarabine, methotrexate, 6-mercaptopril 15-deoxyspergualin, LF15-0195, bredinin, brequinar and / or muromonab-CD3. The calcineurin inhibitor may be cyclosporine A or cyclosporine G. mTor inhibitors include sirolimus, temsirolimus, and zoloft. The anti-CD45 antibody may be an anti-CD45RB antibody. In one embodiment, the immunosuppressant is belatacept.

[0044] The antibody or antigen-binding portion thereof of the present invention and the immunosuppressant drug are can be administered to [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 shows the heavy and light chain variable regions of the 2C10 antibody. The nucleotide sequence shown for the heavy chain (SEQ ID NO: 1) includes a signal peptide (nucleotides 1-57; underlined) and the heavy chain variable sequence (nucleotides 58-396). The corresponding amino acid sequence is shown below (SEQ ID NO: 2), where amino acids 1-19 correspond to the signal sequence (underlined) and amino acids 20-132 correspond to the heavy chain variable region.

[0046] The nucleotide sequence shown for the light chain (SEQ ID NO:3) contains a signal peptide (nucleotide The corresponding nucleotides are the light chain variable sequence (nucleotides 1 to 66; underlined) and the light chain variable sequence (nucleotides 67 to 384). The amino acid sequence is shown below (SEQ ID NO: 4), where amino acids 1-22 are the signal peptide. The amino acids 23 to 128 correspond to the light chain variable region. [Figure 2a] FIG. 2a is a plot showing flow cytometry data confirming binding of 2C10 to human and rhesus CD20+ B cells. [Figure 2b] FIG. 2b is a plot showing CD40 adsorption data from an ELISA assay using various concentrations of 2C10 to confirm binding of 2C10 to human and rhesus CD40, detected with goat anti-mouse IgG-HRP. [Figure 3] Figure 3 is a graph showing dose-dependent inhibition of CD154 binding to human B cells by 2C10. B cells were analyzed for CD154 binding by incubating with histidine-tagged soluble CD154 and analyzing for histidine expression. Results are representative of multiple replicate experiments. [Figure 4] FIG. 4 is a schematic and graph illustrating the principle of the assay involving rhesus or human peripheral blood mononuclear cells (PBMCs) and Jurkat cells. [Figure 5-1]FIG. 5 is a set of graphs showing CD23 expression on CD20+ cells obtained from co-cultures of rhesus PBMCs and Jurkat cells in the presence of various concentrations of 3A8, 5C8, or 2C10 antibodies. [Figure 5-2] FIG. 5 is a set of graphs showing CD23 expression on CD20+ cells obtained from co-cultures of rhesus PBMCs and Jurkat cells in the presence of various concentrations of 3A8, 5C8, or 2C10 antibodies. [Figure 6-1] FIG. 6 is a set of graphs showing CD86 expression on CD20+ cells harvested from co-cultures of human PBMCs and Jurkat cells in the presence of various concentrations of 3A8, 5C8, or 2C10 antibodies. [Figure 6-2] FIG. 6 is a set of graphs showing CD86 expression on CD20+ cells harvested from co-cultures of human PBMCs and Jurkat cells in the presence of various concentrations of 3A8, 5C8, or 2C10 antibodies. [Figure 7-1] FIG. 7 is a set of graphs showing CD23 expression on CD20+ cells of either human or rhesus PBMCs cultured in the presence of either 3A8 or 2C10 antibodies without Jurkat cells. [Figure 7-2] FIG. 7 is a set of graphs showing CD23 expression on CD20+ cells of either human or rhesus PBMCs cultured in the presence of either 3A8 or 2C10 antibodies without Jurkat cells. [Figure 8] Figure 8 is a graph showing peripheral B cell counts in rhesus macaques treated with 2C10, a mouse-rhesus chimeric form modified to contain either rhesus IgG1 (2C10R1) or IgG4 (2C10R4) heavy chain constant regions, and the chimeric IgG1 forms of anti-CD40 3A8 (3A8R1) or anti-CD40 Chi220 (Chi220). [Figure 9]9 is a graph showing T cell-dependent antibody responses in macaque monkeys treated with 2C10R1, 2C10R4, or 3A8R1 antibodies. All animals were immunized with 4-hydroxy-3-nitrophenylacetyl-conjugated keyhole limpet hemocyanin (KLH) after the initial antibody treatment. [Figure 10] Figure 10 is a diagram illustrating the standard macaque model of pancreatic islet allotransplantation. Diabetes was induced in macaque monkeys using streptozotocin. Diabetic monkeys were transplanted with allogeneic islets, and immunosuppression was initiated using basiliximab and sirolimus. Experimental animals received 2C10R4 treatment on days 0 and 7 post-transplant. [Figure 11a] Figure 11a is a plot showing free blood glucose levels (FBG) in four rhesus macaques treated with background immunosuppression and 2C10R4 after islet transplantation. The solid line on the plot represents 2C10 levels in plasma. [Figure 11b] FIG. 11b is a plot showing the FBG of a macaque that received only background immunosuppression. [Figure 12] Figure 12 is a graph showing the results of a competitive blocking assay using human PBMCs incubated with increasing concentrations of 2C10, 3A8, or Chi220 antibodies and stained with APC-conjugated 2C10 to assess the ability of each antibody to cross-block 2C10. [Figure 13] Figures 13a and 13b show sequence alignments of the humanized 2C10 variable regions. Figure 13a: The murine 2C10 VH sequence was aligned to human germline VH1-3 and three humanized sequences, 2C10_h1, 2C10_h2, and 2C10_h3. Figure 13b: The murine 2C10 VL sequence was aligned to human germline VH3-11 and two humanized sequences, 2C10_l1 and 2C10_l2. The 2C10 CDRs are shown in bold. Murine residues in the humanized sequences are underlined. [Figure 14]FIG. 14 shows the amino acid differences in framework 3 between the 2C10HP and 2C10HB1 and 2C10HB2 constructs. [Figure 15] 15 shows the sequences of the heavy and light chain variable regions of humanized 2C10 antibodies, including 2C10HP, 2C10HB1, 2C10HB2, 2C10KP, 2C10KB1, and 2C10KB2. [Figure 16] Figure 16 shows the binding affinity of the humanized 2C10 antibody to CD40 from different primate species. Humanized 2C10 antibody (h2C10) was immobilized on the surface of a CM5 chip by amine coupling. Various concentrations of human, rhesus, and baboon CD40-MBP fusions were analyzed for affinity on a BIACore 3000. Binding affinities were calculated using BIAevaluation software version 4.1.1. [Figure 17] Figure 17 shows that the induction of anti-KLH antibody responses (IgM and IgG) was measured 3 hours after administration of either saline, 10, or 25 mg / kg h2C10 in KLH-immunized monkeys. All control animals demonstrated IgG or IgM antibody responses to the KLH antigen. Individual monkeys treated with 10 mg / kg developed either IgG or IgM antibody responses to KLH, but animals treated with 25 mg / kg 2C10 did not. [Figure 18-1] Whole blood was used for phenotyping of B and T lymphocyte subsets after treatment with 25 mg / kg h2C10 (top row) or 10 mg / kg h2C10 (middle row) or control animals (bottom row). Neither dose of h2C10 had any apparent effect on lymphocyte populations. The lack of discernible B cell depletion was also evident in initial dose-response assessments of primate chimeric forms, including detailed analysis of mature and immature B cell populations. [Figure 18-2]Whole blood was used for phenotyping of B and T lymphocyte subsets after treatment with 25 mg / kg h2C10 (top row) or 10 mg / kg h2C10 (middle row) or control animals (bottom row). Neither dose of h2C10 had any apparent effect on lymphocyte populations. The lack of discernible B cell depletion was also evident in initial dose-response assessments of primate chimeric forms, including detailed analysis of mature and immature B cell populations. [Figure 19] By day 28, humanized 2C10 completely saturated CD40 binding sites on B cells. H2C10 administered in vivo completely blocked the binding of fluorescently labeled 2C10 to B cells. Data show results for humanized 2C10-treated and control monkeys at day 28 after a single dose of 25 mg / kg (top row), 10 mg / kg (middle row), or 0 mg / kg (control; bottom row). Similar results were obtained at days 3, 7, 14, and 21 after infusion. [Figure 20] Mean serum concentrations of h2C10 after treatment of monkeys with either 10 mg / kg or 25 mg / kg up to day 28. Concentrations of 2C10 decline slowly over time, with levels detectable throughout the entire study period. [Figure 21-1] Figures 21a and 21b show the DNA and amino acid sequences of humanized 2C10 (h2C10) in a stabilized IgG4 format. Figure 21a shows the DNA and amino acid sequences of the heavy chain. Figure 21b shows the DNA and amino acid sequences of the light chain. SEQ ID NO: 32: DNA sequence of the heavy chain; SEQ ID NO: 33: amino acid sequence of the heavy chain; SEQ ID NO: 34: DNA sequence of the light chain; SEQ ID NO: 35: amino acid sequence of the light chain. [Figure 21-2] Figures 21a and 21b show the DNA and amino acid sequences of humanized 2C10 (h2C10) in a stabilized IgG4 format. Figure 21a shows the DNA and amino acid sequences of the heavy chain. Figure 21b shows the DNA and amino acid sequences of the light chain. SEQ ID NO: 32: DNA sequence of the heavy chain; SEQ ID NO: 33: amino acid sequence of the heavy chain; SEQ ID NO: 34: DNA sequence of the light chain; SEQ ID NO: 35: amino acid sequence of the light chain. [Figure 21-3]Figures 21a and 21b show the DNA and amino acid sequences of humanized 2C10 (h2C10) in a stabilized IgG4 format. Figure 21a shows the DNA and amino acid sequences of the heavy chain. Figure 21b shows the DNA and amino acid sequences of the light chain. SEQ ID NO: 32: DNA sequence of the heavy chain; SEQ ID NO: 33: amino acid sequence of the heavy chain; SEQ ID NO: 34: DNA sequence of the light chain; SEQ ID NO: 35: amino acid sequence of the light chain. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present disclosure provides anti-CD4 antibodies that may be used in a variety of therapeutic, prophylactic, diagnostic, and other methods. The present invention relates to antibodies and antibody fragments (e.g., antigen-binding portions of the antibodies) that bind to CD40. and blocking the ability of CD40-expressing cells (e.g., B The antibody or fragment thereof of the present invention can achieve this without activating the target cell. may be used to reduce complications associated with organ or tissue transplantation.

[0048] The antibody or antigen-binding portion thereof may be, but is not limited to, a humanized antibody, a human antibody, a monoclonal antibody, or a nucleotide sequence. Clonal antibodies, chimeric antibodies, polyclonal antibodies, recombinantly expressed antibodies, and the foregoing The antigen-binding portion of an antibody includes a portion that specifically binds to CD40. Examples of such antibodies include portions of antibodies that bind to the antibody.

[0049] The present disclosure also provides methods for reducing the likelihood of transplant rejection, treating transplant rejection, and , Compositions and Methods for Inducing Immunosuppression and / or Treating Autoimmune Disorders - Patent application The composition contains an antibody or a fragment thereof that specifically binds to CD40. It is something.

[0050] In one embodiment, the present disclosure provides a composition comprising an antibody (or fragment thereof) of the invention in a mammal. The subject is provided with a method for reducing one or more symptoms of graft-versus-host disease and / or transplant rejection. and administering to a subject an amount sufficient to treat graft-versus-host disease and / or transplant rejection. Provide methods for installing or improving

[0051] In another embodiment, the antibody or antigen-binding fragment is directed against an inflammatory disease or immune disorder, e.g., an autoimmune disorder. The inflammatory or autoimmune disease is caused by CD40-expressing cells. It may be related to.

[0052] In the present invention, an effective amount of an antibody or antigen-binding portion thereof of the present invention is administered to a subject. and methods for reducing the likelihood of transplant rejection in said subject, methods for treating transplant rejection, immunosuppression, Methods for inducing inhibition and / or treating autoimmune disorders are featured and described.

[0053] The disclosure also provides a method for administering to a mammal a composition comprising an antibody of the invention or an antigen-binding portion thereof, administering in an amount sufficient to block a CD40-mediated immune response in a mammal; Also encompassed is a method of blocking CD40 function in a mammal, comprising:

[0054] Another method of the present disclosure relates to inhibiting the growth and / or differentiation of CD40-expressing cells. The method comprises administering an antibody or antigen-binding fragment of the present invention to the cells, When the fragment binds to CD40, the growth and / or differentiation of the cell is inhibited.

[0055] The present disclosure provides a method of treating a subject having a CD40-associated disorder, comprising administering to the subject administering an antibody or antigen-binding fragment of the present invention to a subject, wherein the antibody or antigen-binding fragment inhibits CD4 0 inhibits the growth and / or differentiation of cells in a CD40-associated disorder. The cells may be, but are not limited to, B lymphoblastoid cells, pancreatic cells, lung cells, breast cells, oocyte cells, or the like. colon cells, prostate cells, skin cells, head and neck cells, bladder cells, bone cells or kidney cells It may be a cell.

[0056] The methods of the present invention are directed to the treatment of chronic lymphocytic leukemia, Burkitt's lymphoma, multiple myeloma, T-cell lymphoma, and the like. lymphoma, non-Hodgkin's lymphoma, Hodgkin's disease, Waldenstrom's macroglobulinemia, or can be used to treat Kaposi's sarcoma.

[0057] A further method of the present disclosure includes administering to a subject an effective amount of an anti-CD40 antibody or fragment thereof of the present disclosure. In one embodiment, the method inhibits antibody production by B cells in a subject, comprising administering a fragment thereof to the subject. In this embodiment, the antibody inhibits B cell differentiation and antibody isotype switching in a subject. In another embodiment, the antibody is administered in an amount effective to induce cytotoxicity in the subject. in an amount effective to inhibit the production of inflammatory cytokines and chemokines, and / or T cells and macrophages. The phage is administered in an amount effective to inhibit upregulation of adhesion molecules in the phage. In one embodiment, the antibody is administered in an amount effective to inhibit dendritic cell activation in a subject. It is given.

[0058] In addition to the antibody or fragment thereof of the invention, the method of the invention further comprises administering a second therapeutic agent, e.g. These include immunosuppressants, tumor necrosis factor antagonists (TNF antagonists), CTLA4 antagonists, and anti-IL-6 receptor antagonists. The method may include administering an anti-CD20 antibody, an anti-CD20 antibody, or a combination thereof.

[0059] The antibodies or antigen-binding portions thereof of the present invention may be directed to human CD40 and / or rhesus monkey (rhes us) a compound capable of specifically binding to CD40, for example, recombinant human CD40 and native human CD40 This is what is done.

[0060] As used herein, a CD40-expressing cell is characterized by surface expression of CD40. Any cell, including but not limited to, normal and neoplastic B cells, interdigitating cells, suprabasal cells epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsillar cells and bone marrow-derived cells These are stroma cells.

[0061] humanized antibodies Humanized antibodies of the present disclosure are antibodies derived from a non-human species, in which the non-antigen binding region (and / or The amino acid sequence within the antibody (antigen-binding region) has been altered to make the antibody more similar to a human antibody. Although the antibody has been modified, it still retains its original binding ability.

[0062] The light chain variable region or heavy chain variable region of an antibody consists of three complementary determining regions (CDRs). The CDRs are supported within the variable region by framework regions (FR). In one embodiment, the heavy chain variable region (or light chain variable region) has three CDRs and four CDRs. It contains four framework regions (FRs), which are arranged from the amino terminus to the carboxy terminus. They are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Kabat, EA, et al. Sequences of Pro teins of Immunological Interest,Fifth Ed ition,USDepartment of Health and Human Services,NIH Publication No.91-3242,199 1. Chothia, C. et al., J. Mol. Biol. 196:901-91 7,1987.

[0063] In certain embodiments, a humanized antibody is an antibody molecule derived from a non-human species. One, two, three or all CDRs from the human species and one CDR from a human immunoglobulin molecule The amino acid sequence may comprise one, two, three, four or all of the framework regions.

[0064] The CDRs of the antibodies or antigen-binding portions thereof of the present invention are derived from a non-human source. The fragments of the antibodies or antigen-binding portions thereof of the present invention may also be derived from human sources. Frameworks include human, humanized, and non-human (e.g., modified to reduce antigenicity in humans). modified mouse frameworks) or synthetic frameworks (e.g., consensus sequences) In one embodiment, an antibody of the invention, or an antigen-binding portion thereof, can have at least one The antibody comprises a heavy chain variable region and / or at least one light chain variable region of the antibody.

[0065] Humanized antibodies of the present disclosure can be made by methods known in the art. For example, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Such non-human amino acid residues are often referred to as "import" residues. This is typically taken from an "import" variable domain. ter and coworkers (Jones et al., Nature 321:522- 5,1986;Riechmann et al.,Nature 332:323-7 ,1988;Verhoeyen et al.,Science 239:1534- 6, 1988), the sequences of the hypervariable regions are replaced with the corresponding sequences of a human antibody. Thus, in such humanized antibodies, intact human variable regions are present. Much less than a domain has been replaced with the corresponding sequence from a non-human species. In certain embodiments, humanized antibodies contain residues of the hypervariable regions as well as residues of other variable regions. A human antibody in which at least some of the residues are replaced with residues from analogous positions in a non-human antibody. do.

[0066] Selection of human variable domains (both light and heavy chains) for use in making humanized antibodies According to the "best fit" method, non-human (e.g., mouse) The sequence of the variable domain of a human antibody (e.g., a rodent antibody) is compared with a library of known human variable domain sequences. The human sequences that are closest to the non-human ones are then humanized. Accepted as a human framework for antibodies. See, e.g., Sims et al., JI mmunol.151:2296-308,1993;Chothia et al., See J. Mol. Biol. 196:901-17, 1987. Another method is A specific fragment derived from the consensus sequence of all human antibodies of a given subgroup of light or heavy chains. The same framework is used to create several different humanized antibodies. For example, Carter et al., Proc. Natl. Aca d.Sci.USA 89:4285-9,1992;Presta et al.,J See Immunol. 151:2623-32, 1993.

[0067] Humanized antibodies are made by replacing the variable region sequences that are not directly involved in antigen binding with counterparts derived from human variable regions. The method can be performed by replacing at least one heavy or light chain with a corresponding sequence. Isolating, manipulating and expressing nucleic acid sequences encoding all or part of the variable regions of Sources of such nucleic acids are well known to those skilled in the art, and include, for example, CD40 The humanized antibody or its derivatives can then be obtained from a hybridoma that produces an antibody against the The recombinant DNA encoding the fragment can be cloned into an appropriate expression vector.

[0068] In another example, once a non-human (e.g., murine) antibody is obtained, the variable regions are sequenced. and the positions of CDR and framework residues can be determined. ,et al.(1991)Sequences of Proteins of Im munological interest,Fifth Edition,USD department of Health and Human Services,N IH Publication No.91-3242. Chothia,C.et a l. (1987) J. Mol. Biol., 196:901-917. Light chain variable region and The heavy chain variable region may be ligated to the corresponding constant region. Alternatively, CDR-grafted antibody molecules may be generated by CDR replacement. One, two, three or all of the CDRs of a particular antibody can be replaced. The CDRs may be derived from at least a portion of a non-human animal (e.g., a mouse In some cases, only some CDRs may be replaced (e.g., the CDRs shown in Table 1). The reason is that the CDRs required for the antibody to bind to a predetermined antigen (e.g., CD40) are maintained. Morrison, SL, 1985, Science ,229:1202-1207.Oi et al.,1986,BioTechniq ues,4:214. U.S. Patent No. 5,585,089; U.S. Patent No. 5,225,539; Nos. 5,693,761 and 5,693,762. European Patent No. 519596. J ones et al., 1986, Nature, 321:552-525. Verh oeyan et al., 1988, Science, 239:1534. Beidl er et al., 1988, J. Immunol., 141:4053-4060.

[0069] The antibodies are humanized while retaining high affinity for the antigen and other favorable biological properties. To this end, one example method is The parental sequences and various conceptual humanized products were analyzed using three-dimensional models of the parental and humanized sequences. Humanized antibodies are prepared by the method of analyzing the humanized antibodies. Three-dimensional immunoglobulin models are commonly available. The sequence of a selected candidate immunoglobulin sequence can be predicted. Computer programs are also available that illustrate and display three-dimensional structures. By examining the displayed structures, it is possible to determine the residues involved in the function of the candidate immunoglobulin sequence. Analysis of the possible role of the group, i.e., the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected to produce the desired antibody. The recipe is designed to achieve specific properties (such as high affinity for the target antigen(s)). The nucleotide sequence may be combined with a nucleotide sequence and an import sequence.

[0070] In some embodiments, the humanized anti-CD40 antibody also comprises the constant region of an immunoglobulin ( It also contains at least a portion of a human immunoglobulin. In embodiments, the antibody comprises both a light chain and at least the variable domain of a heavy chain. The antibody optionally comprises heavy chain constant domains CH1, hinge, CH2, CH3, and / or C It may also contain one or more of H4.

[0071] In some aspects of the present disclosure, one or more domains of the humanized antibody are recombinantly expressed. Recombinant expression involves the presence of one or more control sequences, i.e., an operably linked coding sequence. Polynucleotide sequences that are required for expression in a particular host organism may be used. Control sequences suitable for use in prokaryotic cells include, for example, promoters, operating Regulatory sequences in eukaryotic systems include restriction enzymes and ribosome binding site sequences. These include, but are not limited to, promoters, polyadenylation signals and enhancers. Regulatory sequences such as the following are useful for the expression of humanized anti-CD40 antibodies in prokaryotic and eukaryotic host cells: It can be used for the expression and production of antibodies.

[0072] Also disclosed herein are polypeptides containing one, two or all of the CDRs and having at least one other region. At least one different species, for example, but not limited to, human, rabbit, sheep, dog, cat, Cows, horses, goats, pigs, monkeys, apes, gorillas, chimpanzees, ducks, geese, Antibodies or their antigens replaced with sequences from birds, amphibians, reptiles and other animals Binding moieties are also encompassed by this disclosure.

[0073] Human antibodies The human antibodies of the present disclosure were selected from a human-derived phage display library (one or more The Fv clone variable domain sequences are compared to known human constant domain sequences. can be constructed by combining columns (Hoogenboom et al., J. Mo l.Biol.227:381-8,1992;Marks et al.,J.Mol Biol. 222:581-97, 1991). Alternatively, human antibodies can be produced by hybridization. Human myeloma cells for the production of human monoclonal antibodies can be produced by the myeloma method. and mouse-human heteromyeloma cell lines are described, for example, in Kozbor, J. Immunol. .133:3001-5,1984;Brodeur et al., Monoclon al Antibody Production Techniques and Ap applications,pp.51-63(Marcel Dekker,Inc.,N ew York, 1987); and Boerner et al., J. Immunol .147:86-95,1991.

[0074] Upon immunization, they are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. It is also possible to produce transgenic animals (e.g., mice) that express the gene. Homozygous deletion of the antibody heavy-chain joining region (JH) gene in germline mutant mice of Mela. It has been reported that this leads to complete inhibition of endogenous antibody production. Introduction of a human germline immunoglobulin gene array into blastocyst mutant mice results in enhanced antigen stimulation. Stimulation leads to the production of human antibodies. For example, Jakobovits et al. ,Proc.Natl.Acad.Sci.USA 90:2551-5,1993;J akobovits et al.,Nature 362:255-8,1993;B rueggemann et al.,Year Immunol.7:33-40,1 See 993.

[0075] Gene shuffling is also used to derive human antibodies from non-human, e.g., rodent, antibodies. Alternatively, a human antibody may be used, in which case the human antibody will have similar affinity and affinity to the starting non-human antibody. This method (also called "epitope imprinting") ) according to the present invention, the non-human antibody fragments obtained by phage display technology Either the heavy chain variable region or the light chain variable region is placed in a repertoire of human V domain genes. This generates a population of non-human chain / human chain scFv or Fab chimeras. A non-human chain / human chain chimeric scFv or Fab is then isolated, where the human chain contains Antigen binding disrupted upon removal of the corresponding non-human chain from the primary phage display clone The binding site is restored, i.e., the epitope governs the choice of human chain partner (I). Repeating this process to replace the remaining non-human chains results in a humanized Antibodies can be obtained by CDR grafting (see WO 93 / 06213). Unlike traditional humanization of human antibodies, this technique does not replace FR or CDR residues of non-human origin. A fully human antibody free of nucleotides can be obtained.

[0076] Chimeric antibodies A chimeric antibody is a molecule in which different portions are derived from different animal species. The antibody may comprise a variable region derived from a mouse antibody and a constant region of a human immunoglobulin. Antibodies can be produced by recombinant DNA techniques. Morrison, et al. Proc Natl Acad Sci,81:6851-6855(1984). For example, the gene encoding the mouse (or other species) monoclonal antibody molecule can be purified by restriction enzyme digestion. The mouse Fc-encoding region was removed and the human Fc constant region was extracted. The corresponding part of the gene is replaced with the corresponding part of the gene encoding the mouse V region. By recombinant DNA techniques, NA can be ligated to DNA encoding human constant regions. Better et al., Science, 1988, 240 :1041-1043. Liu et al.PNAS,1987 84:3439-3 443. Liu et al., J. Immunol., 1987, 139:3521- 3526. Sun et al. PNAS, 1987, 84:214-218. Nish imura et al., Canc. Res., 1987, 47:999-1005. Wood et al. Nature, 1985, 314:446-449. Shaw et al., J. Natl. Cancer Inst., 1988, 80:1553- 1559. International Patent Applications WO 1987002671 and WO 86 / 01533 . European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 125,023; and U.S. Patent Nos. 173,494 and 4,816,567.

[0077] Variable regions and CDRs The heavy chain variable region, light chain variable region, and The CDRs are shown in Table 1.

[0078] Table 1 SEQ ID NOs: 11 to 31 [Table 1] TIFF0007763612000002.tif228156

[0079] In certain embodiments, the antibody or antigen-binding portion thereof is selected from SEQ ID NOs: 11, 19, 20, 21, 24, 25, and 26, and At least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least At least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80% %, approx. 81%, approx. 82%, approx. 83%, approx. 84%, approx. 85%, approx. 86%, approx. 87%, approx. 88 %, approx. 89%, approx. 90%, approx. 91%, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96 %, about 97%, about 98%, about 99%, or about 100% identical amino acid sequence to the heavy chain variable region It includes the area.

[0080] In certain embodiments, the antibody or antigen-binding portion thereof is selected from SEQ ID NOs: 12, 22, 23, 27, 28, and 29, and at least about the amino acid sequence of the light chain variable region 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, about 98%, about 99%, or about 100% identical amino acid sequence to the light chain variable region. It is something that

[0081] In certain embodiments, the antibody or antigen-binding portion thereof is selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, 40, 41, 4 9, 20, 21, 24, 25, and 26, and At least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, approx. 81%, approx. 82%, approx. 83%, approx. 84%, approx. 85%, approx. 86%, approx. 87%, approx. 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately Heavy chains containing 96%, about 97%, about 98%, about 99%, or about 100% identical amino acid sequences are also acceptable. The variable region and the amino acid sequence of the light chain variable region shown in SEQ ID NOs: 12, 22, 23, 27, 28 and 29. At least about 70%, at least about 75%, at least about 80%, at least about 100% About 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, Approximately 75%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, Approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical amino acid sequence The present invention relates to a method for producing a human LPS comprising administering to a mammalian subject the invention, ... a method for producing a human LPS comprising administering to a mammalian subject the invention, and a light chain variable region comprising the LPS.

[0082] The heavy chain variable region of the antibody or antigen-binding portion thereof comprises the CDRs of the heavy chain variable region of the 2C10 antibody. (CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 13, 14 and 15, respectively) at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 8 0%, approx. 81%, approx. 82%, approx. 83%, approx. 84%, approx. 85%, approx. 86%, approx. 87%, approx. 8 8%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 9 6%, about 97%, about 98%, about 99% or about 100% identical one, two, three or more It may comprise the above complementarity determining regions (CDRs).

[0083] The light chain variable region of the antibody or antigen-binding portion thereof comprises the CDRs of the light chain variable region of the 2C10 antibody. (CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 16, 17 and 18, respectively) at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 8 0%, approx. 81%, approx. 82%, approx. 83%, approx. 84%, approx. 85%, approx. 86%, approx. 87%, approx. 8 8%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 9 6%, about 97%, about 98%, about 99% or about 100% identical one, two, three or more It may comprise the above CDRs.

[0084] The heavy chain variable region of the antibody of the present invention or the antigen-binding portion thereof may be the heavy chain variable region of the 2C10 antibody. CDRs (CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 13, 14, and 15, respectively) and at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 95%, at least about 99%, about 70%, about 75% , about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87% , about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95% , about 96%, about 97%, about 98%, about 99% or about 100% identical one, two, three or and the antibody or antigen-binding portion thereof may comprise one or more complementarity-determining regions (CDRs). The light chain variable region of the 2C10 antibody is composed of the CDRs of the light chain variable region of the 2C10 antibody (SEQ ID NOs: 16 and 17, respectively). 7, 18) and at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately It may contain one, two, three or more CDRs that are 99% or about 100% identical. do.

[0085] The heavy chain variable region of the antibody or antigen-binding portion thereof comprises the CDRs of the heavy chain variable region of the 2C10 antibody. (CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 13, 14 and 15, respectively) at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 8 0%, approx. 81%, approx. 82%, approx. 83%, approx. 84%, approx. 85%, approx. 86%, approx. 87%, approx. 8 8%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 9 6%, about 97%, about 98%, about 99% or about 100% identical CDRs. It is possible.

[0086] In one embodiment, the light chain variable region of the antibody, or antigen-binding portion thereof, is the light chain variable region of the 2C10 antibody. The CDRs of the variable regions (CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 16, 17 and 18, respectively) CDR3) and at least about 70%, at least about 75%, at least about 80%, or at least Also about 85%, at least about 90%, at least about 95%, at least about 99%, about 70% , about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86% , about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% , about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical It contains R.

[0087] In one embodiment, the heavy chain variable region of the antibody or antigen-binding portion thereof comprises the heavy chain variable region of the 2C10 antibody. The CDRs of the variable regions (CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 13, 14 and 15, respectively) CDR3) and at least about 70%, at least about 75%, at least about 80%, or at least Also about 85%, at least about 90%, at least about 95%, at least about 99%, about 70% , about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86% , about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% , about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical R, and the light chain variable region of the antibody or antigen-binding portion thereof comprises the light chain variable region of the 2C10 antibody. The CDRs of the chain variable regions (CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 16, 17 and 18, respectively) and CDR3) and at least about 70%, at least about 75%, at least about 80%, At least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 70% %, approx. 75%, approx. 80%, approx. 81%, approx. 82%, approx. 83%, approx. 84%, approx. 85%, approx. 86 %, approx. 87%, approx. 88%, approx. 89%, approx. 90%, approx. 91%, approx. 92%, approx. 93%, approx. 94 %, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical This includes DR.

[0088] In certain embodiments, the heavy chain variable region of the antibody, or antigen-binding portion thereof, is The CDRs of the heavy chain variable region of the antibody (CDR1, CDR2, CDR3 shown in SEQ ID NOs: 13, 14, and 15, respectively) and the antibody or its antigen-binding domain contains three CDRs identical to those of the CDRs (CDR2 and CDR3), The light chain variable region of the fragment is a CDR of the light chain variable region of the 2C10 antibody (SEQ ID NO: 16). , 17, 18) containing the same three CDRs as is.

[0089] The present disclosure provides a method for producing a medicament comprising administering to a subject the heavy chain variable region and the light chain variable region of antibody 2C10, respectively. (SEQ ID NO: 11) and light chain variable region (SEQ ID NO: 12) at least about 70%, At least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least About 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82% %, approx. 83%, approx. 84%, approx. 85%, approx. 86%, approx. 87%, approx. 88%, approx. 89%, approx. 90 %, approx. 91%, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96%, approx. 97%, approx. 98 %, about 99% or about 100% identical amino acid sequence to the antibody.

[0090] In a related embodiment, the anti-CD40 antibody, or antigen-binding portion thereof, is, for example, 2C10 It contains the CDRs of the heavy chain variable region and / or the light chain variable region.

[0091] In one embodiment, the antibody or antigen-binding portion thereof comprises the heavy chain variable region and light chain variable region of the 2C10 antibody. The heavy and light chain variable regions are identical to those of the heavy and light chain variable regions (SEQ ID NO: 11 and SEQ ID NO: 12, respectively). It contains a variable region.

[0092] In various embodiments, the antibody or antigen-binding portion thereof is an antibody that binds to the 2C10 antibody. Those that specifically bind to an epitope that overlaps with the epitope bound by the 2C10 antibody. At least about 70%, at least about 75%, at least about 80%, at least about About 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, Approximately 75%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, Approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical epitopes The epitope specifically binds to the epitope present in the sequence of SEQ ID NO: 6. or may be present within the sequence of amino acids 8 to 40 of SEQ ID NO: 6. could be In certain embodiments, the CDRs corresponding to those in Table 1 have sequence variations. For example, 1, 2, 3, 4, 5, 6, 7 or 8 residues of the CDRs or Less than 20%, less than 30%, or less than about 40% of all residues are substituted or deleted The CDRs can be present in an antibody (or antigen-binding portion thereof) that binds to CD40.

[0093] Antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted, or added are also included in the present invention. Such modifications may have an adverse effect on the biological properties of the peptide, such as binding activity. For example, an antibody may have a function to modify binding to an antigen, but not have a substantial effect on the In another example, the amino acid sequence may have amino acid substitutions in the framework regions to improve the , a selected small number of acceptor framework residues are replaced with the corresponding donor amino acids. The donor framework may be a mature or germline human antibody framework. The amino acid sequence may be a sequence or a consensus sequence. For guidance on how to do this, see Bowie et al., Science, 2002. 47:1306-1310(1990).Cunningham et al., Sci. ence,244:1081-1085(1989).Ausubel(ed.), Cu rrent Protocols in Molecular Biology,Joh n Wiley and Sons, Inc. (1994). T. Maniatis, E. .F.Fritsch and J.Sambrook,Molecular Clon ing:A Laboratory Manual,Cold Spring Harb or laboratory,Cold Spring Harbor,NY(19 89).Pearson,Methods Mol.Biol.243:307-31( 1994).Gonnet et al.,Science 256:1443-45( 1992).

[0094] The peptides of the present invention may be, for example, less than about 30%, about 25%, about 20%, about 15%, about 10%, or less than about 30%. %, about 5%, or about 1% of the amino acid residues are substituted or deleted but are essentially the same. The antibodies described herein retain the same immunological properties, including but not limited to, binding to CD40. It may be a functionally active variant of the disclosed antibodies or antigen-binding portions thereof.

[0095] The antibody or antigen-binding portion thereof has biological activity, e.g., binding to an antigen such as CD40. Peptide variants, peptide analogs, peptide orthologues, and peptide homologues and peptide derivatives may also be included. The peptide may contain one or more amino acid analogs ( For example, non-naturally occurring amino acids, amino acids that occur naturally only in unrelated biological systems. , modified amino acids derived from mammalian systems, etc., and those with substituted bonds. It can be a peptide as well as other modifications known in the art.

[0096] The antibody or antigen-binding portion thereof may be derivatized or linked to another functional molecule. For example, an antibody may be linked to one or more other molecular entities, such as another antibody, a detectable agent, Immunosuppressants, cytotoxic agents, pharmaceutical agents, proteins or other molecules that can mediate association with other molecules. The peptide (e.g., streptavidin core region or polyhistidine tag), amino acid sequence amino acid linkers, signal sequences, immunogenic carriers, or ligands useful for protein purification, For example, glutathione-S-transferase, histidine tag, and Staphylococcus aureus promoter can be operably linked to protein A (by chemical coupling, genetic fusion, non-covalent bonding) Cytotoxic agents include radioisotopes, chemotherapeutic agents, and toxins, e.g. For example, enzymatically active toxins of bacterial, fungal, plant or animal origin, as well as fragments thereof, may be mentioned. Such cytotoxic agents can be coupled to the humanized antibodies of the present disclosure using standard procedures, e.g., For example, it can be used to treat patients for whom treatment with the antibody is indicated.

[0097] One type of derivatized protein is two or more proteins (of the same type or different types). Suitable cross-linking agents include heterobifunctional and has two different reactive groups separated by a suitable spacer ( For example, m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homo Bifunctional ones (e.g., disuccinimidyl suberate) are also included. Useful detectable agents with which proteins can be derivatized (or labeled) include fluorescent agents, species Examples include various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting exemplary fluorescent detectable agents include fluorescein, fluorescein-1, sothiocyanate, rhodamine, and phycoerythrin. Alternatively, the antibody may be coupled to a detectable enzyme, such as alkaline phosphatase, horseradish pea, or the like. peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase Proteins may also be derivatized with prosthetic groups (e.g., It may also be derivatized with streptavidin / biotin and avidin / biotin.

[0098] In another embodiment, the humanized anti-CD40 antibody or fragment thereof is used without labeling, and the humanized anti-CD40 antibody or fragment thereof is Detection is performed using a labeled antibody that binds to the CD40 antibody or a fragment thereof.

[0099] antibody fragment The antibody may be full-length, or may be a fragment or fragments of the antibody that contain the antigen-binding portion. including, but not limited to, Fab, F(ab')2, Fab', F(ab)' , Fv, single-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tr i-scFv), Fd, dAb fragments (e.g., Ward et al., Nature, 341:544-546 (1989)), isolated CDRs, diabodies, triabodies, tetrabodies Multispecific antibodies formed from antibody fragments, linear antibodies, single-chain antibody molecules, and antibody fragments. Alternatively, antibody fragments may be linked together using recombinant or synthetic linkers. Single chain antibodies made from the same are also encompassed by the present disclosure. Bird et al. Science ,1988,242:423-426. Huston et al.,Proc.Nat l.Acad.Sci.USA,1988,85:5879-5883.

[0100] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments , each containing one antigen-binding site, the remainder being the "Fc" fragment, the name given to the fragments that are easily crystallized. Pepsin treatment produces F(ab')2 fragments, which are fragments of 2 It has two antigen-binding sites and still has the ability to cross-link antigens.

[0101] An Fv is the minimum antibody fragment that contains a complete antigen-binding site. A two-chain Fv species comprises one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. Single-chain Fv (scFv) species consist of a dimer of one heavy chain variable domain. The light and heavy chains are connected by a flexible peptide linker. The two chains can be covalently linked so as to associate in a "dimeric" structure similar to that of a two-chain Fv species. The three CDRs of each variable domain interact to form a V H -V L Antigen binding on the surface of the dimer It is in this configuration that the six CDRs together define the antigen-binding site in the antibody. However, a single variable domain (i.e., three domains specific for an antigen) Although even a single Fv (containing only the CDRs) has the ability to recognize and bind to an antigen, The affinity is lower than that of the normal binding site.

[0102] The Fab fragment contains the heavy chain variable domain and the light chain variable domain, and also contains the light chain constant domain. The Fab' fragment contains the Fab' domain and the first constant domain (CH1) of the heavy chain. , which has several residues added to the carboxyl terminus of the heavy chain CH1 domain and forms the hinge region of the antibody. Fab'- differs from Fab fragments by the inclusion of one or more cysteines in the Fab'- region. SH indicates that the cysteine ​​residue(s) in the constant domain(s) bear a free thiol group. F(ab')2 antibody fragments originally have hinge cysteines between them. These antibodies are produced as Fab' fragment pairs, which are also used for other chemical couplings of antibody fragments. It is also known as

[0103] Single-chain Fv or scFv antibody fragments are H and V L domains, where these The domains are present in a single polypeptide chain. Generally, an scFv polypeptide further comprises To, V H Domains and V L It contains a polypeptide linker between the domains, which allows cFv can form the desired structure for antigen binding. For example, see Pluckthuen, The Pharmacology of M onoclonal antibodies,vol.113,Rosenburg a nd Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0104] Diabodies are antibody fragments with two antigen-binding sites, and the fragments contain heavy chain variable domains. In (V H ) is the light chain variable domain (V L ) linked within the same polypeptide chain (V H -V L ) to allow pairing between these two domains on the same chain. By using a linker that is too short, the domains will not pair with the complementary domains of another strand. Diabodies are bivalent or bispecific, meaning that two antigen-binding sites are created. Diabodies may be used as described, for example, in European Patent No. 404,097; International Publication No. WO 1993 / 013499; No. 01161;Hudson et al., Nat.Med.9:129-34,20 03; and Hollinger et al., Proc. Natl. Acad. Sci USA 90:6444-8, 1993. Abodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129 -34,2003.

[0105] Antibody fragments can be produced by conventional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies. Smaller fragments allow for more rapid clearance, resulting in improved access to solid tumors. For a review of some specific antibody fragments, see Hudson et al. See I. Nat. Med. 9:129-134, 2003.

[0106] Various techniques have been developed for the production of antibody fragments. Traditionally, such fragments have been produced by: Derived by proteolytic digestion of intact antibodies (e.g., Mori oto et al., J.Biochem.Biophys.Methods 24: 107-17, 1992; and Brennan et al., Science 229 However, such fragments are now widely used in recombinant host cells. Fab, Fv and ScFv antibody fragments can all be produced directly by the It can be expressed in E. coli and secreted, thus producing large amounts of this Such fragments can be readily produced from antibody phage libraries. Alternatively, the Fab'-SH fragment may be isolated from Escherichia coli (E. coli). oli) and chemically coupled to form F(ab')2 fragments. (Carter et al.,Bio / Technology 10:163-7,1 In another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fa has a long in vivo half-life and contains salvage receptor binding epitope residues. F(ab')2 and F(ab')2 fragments are described in U.S. Patent No. 5,869,046. Other techniques for making strips will be apparent to those skilled in the art.

[0107] The antibodies of the invention, or antigen-binding portions thereof, comprise at least one constant domain, e.g., (a) I (b) the constant domain of IgA; etc.

[0108] Any antibody isotype, e.g., IgG (e.g., IgG1, IgG2, IgG 3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE are disclosed herein. The antibody or antigen-binding portion thereof may be a mammalian (e.g., murine, human) antibody or may be the antigen-binding portion thereof. The light chains of the antibody may be of the kappa or lambda type. Humanized anti-CD40 antibodies are derived from more than one immunoglobulin class or isotype. The nucleotide sequences may include sequences that are specific for optimizing the desired effector function. The choice of domain is within the ordinary skill in the art.

[0109] The antibodies or antigen-binding portions thereof of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific or bispecific antibodies or fragments thereof may be specific to one target. The antibodies may be specific for different epitopes of a target polypeptide (e.g., CD40). , antigen-binding domains specific for more than one target polypeptide (e.g., CD40 and and other antigen-binding domains specific for antigens associated with transplant rejection or autoimmune disease). In one embodiment, the multispecific antibody or antigen-binding portion thereof may comprise at least Each of the variable domains comprises two different variable domains, each of which binds to a different antigen. Alternatively, they may specifically bind to different epitopes on the same antigen. al.,1991,J.Immunol.147:60-69.Kufer et al. ., 2004, Trends Biotechnol. 22:238-244. The antibody may be linked to another functional molecule, such as another peptide or protein, or For example, the antibody or fragment thereof may be co-expressed with one or more other molecular entities, For example, by operably linking (e.g., chemical coupling, genetic fusion) to another antibody or antibody fragment. In some cases, the bispecific antibody may be attached to a second binding specificity (by non-covalent association or other means). Monospecific or multispecific antibodies may be generated. For example, the present disclosure includes antibodies directed to one or more of the immunoglobulins. One arm of the immunoglobulin is specific for CD40, and the other arm of the immunoglobulin is specific for a second therapeutic target. or conjugated to a therapeutic moiety, e.g., an immunosuppressant drug. Multispecific antibodies are included.

[0110] Antibody production The present disclosure provides a method for producing an antibody or antigen-binding portion thereof that specifically binds to CD40. The present invention provides a method for:

[0111] For example, a non-human animal may be immunized with a composition containing CD40, and specific antibodies may then be generated in the animal. The method further comprises assessing binding of the antibody to CD40. That's fine.

[0112] In one embodiment, the present disclosure provides a hybridoma expressing an antibody that specifically binds to CD40. The present invention provides a method for producing domas, the method comprising the steps of: transfecting an animal with CD40 or immunizing the animal with a composition comprising the fragment; isolating splenocytes from the animal; and producing hybridomas that produce antibodies that specifically bind to CD40. This involves selecting hybridomas. Kohler and Milstei n, Nature, 256:495, 1975. Harlow, E. and Lane, D.Antibodies:A Laboratory Manual,Cold Sp ring Harbor Laboratory Press,Cold Spring Harbor, NY, 1988.

[0113] In one embodiment, CD40 is used to immunize mice intraperitoneally or intravenously. One or more booster immunizations may be given, or none may be given. , e.g., ELISA (enzyme-linked immunosorbent assay) or flow cytometry Mice with sufficient anti-CD40 antibody titers are used for fusion. Mice may or may not be boosted with antigen 3 days before being sacrificed and the spleen removed. Mouse splenocytes are isolated and fused with a mouse myeloma cell line using PEG. The resulting hybridomas are then screened for the production of antigen-specific antibodies. The cells are plated and then incubated in a selective medium. Supernatants from the samples were screened for human anti-CD40 monoclonal antibodies by ELISA. Antibody-secreting hybridomas are replated and screened again. If still positive for anti-CD40 monoclonal antibodies, It can be subcloned.

[0114] Adjuvants that can be used to enhance the immunogenicity of CD40 include peptides or Any agent(s) that acts to enhance the immune response to the peptide combination Non-limiting examples of adjuvants include alum, aluminum phosphate, , aluminum hydroxide, MF59 (4.3% w / v squalene, 0.5% w / v poly Sorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (S pan 85), CpG-containing nucleic acid, QS21 (saponin adjuvant), MPL (mono Phosphoryl lipid A), 3DMPL (3-O-deacylated MPL), Aquilla extract ISCOMS (e.g., Sjolander et al. (1998) J. Leu kocyte Biol.64:713;WO 90 / 03184;WO 9 6 / 11711;WO 00 / 48630;WO 98 / 36772;Country International Publication No. 00 / 41720; International Publication No. 06 / 134423 and International Publication No. 07 / 0 26190), LT / CT variants, poly(D,L-lactide-co-glycolide) ( PLG) microparticles, Quil A, interleukin, Freund's, N-acetyl- Muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl- Nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP) N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl N-2-(1'-2'-diphosphate-aluminum-3-hydroxyphosphate) (folyloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI (which is a mixture of three components extracted from bacteria: monophosphoryl lipid A, Trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) with 2% squalene / Tween 80 emulsion).

[0115] The animal to be immunized may be any animal that can produce recoverable antibodies upon administration of an immunogen. Including but not limited to rabbits, mice, rats, hamsters, goats, horses, monkeys, baboons and rhesus monkeys In one embodiment, the host is transgenic and produces human antibodies. For example, mice expressing segments of human immunoglobulin genes. No. 8,236,311; No. 7,625,559 and No. 5,770,429 ( (The disclosure of each of these is incorporated herein by reference in its entirety.) rg et al., Nature 368(6474):856-859, 1994. Lonberg, N., Handbook of Experimental Phar. Macology 113:49-101,1994. Lonberg, N. and H. uszar,D.,Intern.Rev.Immunol.,13:65-93,19 95. Harding, F. and Lonberg, N., Ann. NYAcad. .Sci.,764:536-546,1995.

[0116] The antibodies or portions thereof of the present invention encode the light and heavy chains (or portions thereof) of the desired antibody. The antibody (or antibodies) can be produced by host cells transformed with DNA encoding the antibody. The cells (or a portion thereof) may be isolated and / or purified from such culture supernatants and / or cells using standard techniques. For example, the host cell may encode the antibody light chain, heavy chain, or both, and may be purified. It is also possible to use recombinant DNA techniques to transform either the light or heavy chains. a portion of the DNA encoding at least a portion of either or both that is not necessary for binding The entire sequence may be removed, for example the constant region.

[0117] The present invention also provides at least one antibody of the present invention that specifically binds to CD40 or a The present invention also encompasses nucleic acids or polynucleotides encoding the antigen-binding portion of the nucleic acid. The isolated antibodies of the present disclosure may be expressed in cells to produce the antibodies or antigen-binding portions thereof. The nucleic acid or polynucleotide is at least about 7'-nucleotides having the same sequence as any one of SEQ ID NOs: 11 to 29. 0%, at least about 75%, at least about 80%, at least about 85%, at least about 9 0%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 8 1%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 8 9%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 9 7%, about 98%, about 99% or about 100% identical peptides It contains two sequences.

[0118] In the present invention, a nucleic acid sequence having at least about 70% or at least one of SEQ ID NOs: 11 to 29 is also included. About 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, Approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, Approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, At least one nucleic acid or polypeptide encoding about 99% or about 100% identical peptide Expression vectors containing the oligonucleotides are described.

[0119] Also, a functionally active variant of the antibody of the present invention or an antigen-binding portion thereof is encoded. Such nucleic acid molecules may be used in conjunction with any antibody or its derivatives of the present invention. Nucleic acids encoding antigen-binding portions and nucleic acids encoding medium stringency, high stringency or can hybridize under very high stringency conditions. Guidance for performing this is available in Current Protocols in Molecules. ar Biology,John Wiley & Sons,NY6.3.1-6 See, e.g., .3.6, 1989, which is incorporated herein by reference. The specific hybridization conditions referred to in the document are as follows: (1) medium stringency; Hybridization conditions: 6x SSC at approximately 45°C, followed by 0.2x SSC, 0.1% (2) one or more washes in SDS at 60°C; (3) high stringency hybridization Conditions: 6×SSC at approximately 45°C, followed by 0.2×SSC, 0.1% SDS at 65°C one or more washes; and (3) ultra-high stringency hybridization conditions: 65 0.5 M sodium phosphate, 7% SDS at 65°C, followed by 0.2× SSC, 1% SDS at 65°C Wash at least once.

[0120] Nucleic acids or polynucleotides encoding the antibodies or antigen-binding portions thereof of the invention can be used in any suitable The antibody or its derivatives are introduced into an expression vector that can be expressed in a suitable expression system, and then the expressed antibody or its derivatives are The antibodies of the present invention or antigen-binding portions thereof can be isolated or purified. The loaded nucleic acid may be translated in a cell-free translation system. issue. Queen et al., Proc Natl Acad Sci USA, 86 :10029-10033(1989).

[0121] The nucleic acids of the invention can be expressed in a variety of suitable cells, including prokaryotic and eukaryotic cells, e.g., For example, bacterial cells (e.g. E. coli), yeast cells, plant cells, insect cells and Expression can be achieved in mammalian cells, including those of the human genome. Several mammalian cell lines are known in the art. Known for the American Type Culture Collection Non-limiting examples of such cells include immortalized cell lines available from the American College of Cancer (ATCC). , any cell line of mammalian origin or mammalian-like characteristics, including but not limited to, monkey kidney Hepatocytes (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney liver (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NS0, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, H eLa, Madin-Darby bovine kidney (MDBK), parental cell line for myeloma and lymphoma cells; Derivatives and / or modified variants are also included. Modified variants include, for example, glycoproteins. These include modified can profile and / or site-specific integration site derivatives.

[0122] The disclosure also provides a cell comprising a nucleic acid described herein. The antibody of the present invention or its antigen-binding domain may be a doma or a transfectant. The moieties can be expressed in a variety of cells, the types of cells being discussed herein. There are.

[0123] For example, when using recombinant techniques to generate a humanized antibody or antigen-binding portion thereof, The antibody or portion thereof may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. When antibodies are produced intracellularly, the first step is to allow the cells to release the protein. Particulate debris, either host cells or lysed fragments, can be disrupted to allow for the release of It can be removed by centrifugation or ultrafiltration. Carter et al., 1992, Bio / Technology 10:163-167 reports on the E. coli A procedure has been described for isolating antibodies secreted into the periplasmic space. The yeast was treated with sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride. The cells are thawed in the presence of PMSF for approximately 30 minutes. Cell debris is removed by centrifugation. When the antibody is secreted into the medium, the supernatant of such an expression system is first removed by Commercially available protein concentration filters, such as Amicon or Millipore Pel The antibody can be concentrated using a Licon ultrafiltration unit. A variety of methods can be used.

[0124] The antibody or portion thereof prepared in the cells can be purified by, for example, hydroxylapatite chromatography. Purified using filtration, gel electrophoresis, dialysis and affinity chromatography Affinity chromatography is a typical purification method. The suitability of all Protein A molecules depends on the immunoglobulin Fc domain (if any) present in the antibody. Protein A is based on human γ1, γ2, or γ4 heavy chains, depending on the species and isotype. can be used to purify antibodies from the 983 J. Immunol. Meth. 62:1-13). Protein G is Recommended for all mouse isotypes and human γ3 (e.g., Guss et al., 1986 EMBO J.5:1567-1575). The matrix is ​​most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene Zen allows for faster flow rates and shorter processing times than can be obtained with agarose. The antibody is C H3 If it includes the domain, Bakerbond ABX.TM. Resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody to be recovered, other techniques for protein purification, such as ion exchange, may also be used. fractionation on a conversion column, ethanol precipitation, reversed-phase HPLC, chromatography on silica, Chromatography on Parin Sepharose™, anion or cation exchange resin ( Chromatography on e.g. polyaspartic acid columns, chromatofocusing , SDS-PAGE, and aluminum sulfate precipitation are also available.

[0125] After the preliminary purification step(s), if any, the mixture containing the antibody of interest and contaminants is In this case, an elution buffer with a pH of about 2.5 to 4.5 is used, typically with a low salt concentration (e.g., It is subjected to low pH hydrophobic interaction chromatography performed at approximately 0 to 0.25 M salt. obtain.

[0126] Hybridomas or other cells that produce antibodies that bind to CD40, preferably with high affinity. The cells can then be subcloned and further characterized. One clone from the 1000 cells that retained the reactivity of the parental cells (by ELISA) was then They can be selected for the generation of cell banks and for antibody purification.

[0127] Alternatively, the antibodies or antigen-binding portions thereof of the present invention can be produced using methods well known in the art. Solid Phase Peptide Synthesis sis:A Practical Approach by E.Atherton a nd RCSheppard, published by IRL at Oxfo rd University Press (1989). Methods in Mol ecular Biology,Vol.35:Peptide Synthesis Protocols(ed.MWPennington and BMDunn ),chapter 7.Solid Phase Peptide Synthesis s, 2nd Ed., Pierce Chemical Co., Rockford, I L (1984). G.Barany and RBMerryfield,The Peptides:Analysis,Synthesis,Biology,edit ors E.Gross and J.Meienhofer,Vol.1 and V ol.2, Academic Press, New York, (1980), pp.3 -254. M.Bodansky,Principles of Peptide Sy nthesis, Springer-Verlag, Berlin (1984).

[0128] Additional antibodies against the CD40 epitope recognized by 2C10 (e.g., monoclonal antibodies) Antibodies (clonal, polyclonal, polyspecific or monospecific antibodies) can be used, for example, in the production of antibodies. In one example, the 2C10 antibody can be used to generate a nucleotide sequence that is recognized by the 2C10 antibody. The coding sequence of the epitope to be identified was determined by binding to glutathione S-transferase (GST). It is expressed as a C-terminal fusion (Smith et al., Gene 67:31-4 0, 1988). This fusion protein was purified with glutathione-Sepharose beads. , eluted with glutathione, cleaved with thrombin (at a modified cleavage site), and Purified for immunization of herons. Primary immunization was performed using complete Freund's adjuvant. Subsequent immunizations are performed with incomplete Freund's adjuvant. Western blot and immunoprecipitation analysis (thrombin cleavage of GST fusion protein) Immune serum is monitored by CNBr-Sepharose binding. The specificity of the antiserum is determined by the binding of the GS1 protein to a group of unrelated GS1 proteins. It can be measured using T protein.

[0129] As an alternative or adjunct immunogen to GST fusion proteins, the polypeptides of the present invention may be used in relatively solid state immunogens. A peptide corresponding to the immunogenic region of the protein was prepared and attached to keyhole limpet hemocyanin (KLH). Each of these peptides may be coupled via an introduced C-terminal lysine. Antisera against the antibody were similarly affinity purified with peptides conjugated to BSA. The specificity was confirmed by ELISA or Western blot analysis (peptide conjugates or Western blot or immunoprecipitation (expressed as a GST fusion protein) The test is performed by the following method (using a polypeptide containing the same polypeptide).

[0130] Alternatively, a CD40 antibody that specifically binds to the CD40 epitope recognized by the 2C10 antibody may be used. Monoclonal antibodies may be prepared using standard hybridoma technology (e.g., ,Kohler et al.,Nature 256:495-7,1975;Koh ler et al.,Eur.J.Immunol.6:511-9,1976;Ko hler et al.,Eur.J.Immunol.6:292-5,1976;H ammerling et al., Monoclonal Antibodies a nd T Cell Hybridomas, Elsevier, NY, 1981) Once generated, monoclonal antibodies will also be tested for specific recognition by Western blot. Alternatively, the monoclonal antibody may be tested by immunoprecipitation analysis. can be prepared using the polypeptides and phage display libraries of the present invention (V aughan et al., Nat. Biotechnol. 14:309-14,1 996).

[0131] The epitope fragments are synthesized by standard techniques, e.g., PCR and inserted into pGEX expression vectors. The fusion protein may be produced by cloning a fragment of ) and purified using a glutathione agarose affinity matrix. To minimize the possibility of low affinity or specificity of the antisera being an issue, Two or three such fusions were generated for each fusion, and each fusion was transferred to at least two rabbits. Antisera are raised by a series of injections, e.g., at least three booster injections. may be included.

[0132] For large-scale, low-cost production of polyclonal antibodies, appropriate animal species must be selected. Polyclonal antibodies can be isolated, for example, from the milk or colostrum of immunized cows. Bovine colostrum contains 28g of IgG per liter, while bovine milk The juice contains 1.5 g of IgG per liter (Ontsouka et al., J. Dairy Sci. 86: 2005-11, 2003). Clonal antibodies can also be isolated from the yolk of eggs from immunized chickens (Sar ker et al.,J.Pediatr.Gastroenterol.Nutr. 32:19-25,2001).

[0133] Assay The antibody or antibodies may be used to confirm their specificity to the antigen of interest and / or to test their properties. A variety of methods can be used to assay the antigen-binding portion of a protein. An example of how this can be done is the serum screening method described in U.S. Patent Application Publication No. 2004 / 0126829. Anti-CD40 antibodies are used in a variety of assays known for binding to CD40. They can be characterized by a variety of techniques, for example, ELISA, microtiter plate The cells were coated with CD40 or CD40 fragments (in PBS) and then incubated with unrelated proteins. Block with a protein such as bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from mice immunized with CD40 (or solutions containing anti-CD40 antibodies) is added to each well and incubated. The plate is washed and then an enzyme (e.g., Incubate with a secondary antibody conjugated to alkaline phosphatase After washing, the plate is developed with the enzyme's substrate (e.g., ABTS) and a specific OD In another embodiment, the selected monoclonal antibodies bind to unique epitopes. To determine whether the antibody binds, the antibody can be biotinylated, which can then be used in streptavidin. The anti-CD40 antibody can be detected using an avidin-labeled probe. may be tested for by Western blotting.

[0134] The antibodies of the present disclosure, or antigen-binding fragments, variants, or derivatives thereof, also have the ability to bind to antigens. The affinity of an antibody for an antigen may be described or specified in terms of the binding affinity of the antibody to the antigen. can be measured experimentally using various methods (e.g., Berzofsky et al., “Antibody-Antigen Interactions,”In Funda mental Immunology,Paul,WE,Ed.,Raven Pr. ess:New York,NY(1984);Kuby,Janis Immun ology,WHFreeman and Company:New York,N , Y. (1992); and methods herein). Compatibility measurements may differ when measured under different conditions (e.g., salt concentration, pH). Therefore, affinity and other antigen binding parameters (e.g., K D , K. a , K. d Measurement of the antibody and antigen activity is preferably carried out using standardized solutions and standardized buffers. It can be done.

[0135] The antibodies of the present invention, or antigen-binding portions thereof, bind to CD40 in a concentration of approximately 10 -7 Less than M, about 10 -8 M not yet Full, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M , about 10 -7 M ~ about 10 -12 M, about 10 -8 M ~ about 10 -11 M, about 10 -9 M ~ approx. 1 0 -10 M or about 10 -8 M ~ about 10 -12 Dissociation constant of M (K D ) that binds specifically to That is why.

[0136] Also, the ability of the antibody (or fragment thereof) to block binding of CD40 to CD154. or using assays to test the ability to inhibit or reduce CD40-mediated responses. It may also be used.

[0137] As used herein, the terms "inhibit binding" and "block binding" (e.g., The terms "inhibition / blocking of CD154 binding to CD40" are used interchangeably and partially inhibit It includes both harm / blockage and complete inhibition / blockage. In addition, inhibition and blockage are , when contacted with an anti-CD40 antibody or portion thereof disclosed herein. Any binding of CD154 to CD40 compared to ligand not in contact with the body a measurable reduction in, for example, at least about 10%, 20% of CD154 relative to CD40 , 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 10 It is also intended to include 0% blocks.

[0138] In one embodiment, the activation or inhibition of B cells is achieved by activating or inhibiting CD23, CD80, CD86, and One or more markers selected from any additional suitable markers on CD20+ cells This can be measured by measuring the expression of

[0139] The antibodies or fragments thereof of the present invention are characterized by their effect on T cell-mediated antibody responses. For example, the antibody or fragment thereof can be administered to a mammal by administering the antibody or antigen-binding portion thereof to the mammal. 1mg / kg body weight to approximately 50mg / kg body weight, approximately 2mg / kg body weight to approximately 40mg / kg body weight , approx. 3mg / kg body weight ~ approx. 30mg / kg body weight, approx. 5mg / kg body weight ~ approx. 20mg / kg Dosage ranges from about 8 mg / kg body weight to about 13 mg / kg body weight, and about 1 mg / kg body weight , about 2 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 15 mg / kg Body weight, approx. 20mg / kg body weight, approx. 25mg / kg body weight, approx. 30mg / kg body weight, approx. 35m g / kg body weight, approximately 40 mg / kg body weight, approximately 50 mg / kg body weight, approximately 60 mg / kg body weight, When administered at about 70 mg / kg body weight or about 80 mg / kg body weight, I It is capable of inhibiting the production of gM and / or IgG.

[0140] The antibody or fragment thereof of the present invention is characterized by its effect on prolonging graft survival after transplantation. The antibody or fragment thereof of the present invention may be administered alone or in combination with one or more other immunosuppressants. In combination with this, graft survival was increased to approximately 30%, 40%, 50%, 60%, 70%, and 80%, approximately 90%, approximately 2 times, approximately 5 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 30 times times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times, approximately 55 times, approximately over 40%, approximately over 50%, approximately More than 60%, more than about 70%, more than about 80% or more than about 90%, more than about 2 times, more than about 5 times, more than about 10 times, The prolongation may be about 20 times, about 30 times, or about 40 times. The antibody or fragment thereof may prolong the survival of pancreatic islet allografts.

[0141] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention: a) bind to CD154; c) capable of blocking the binding of CD40 to antigen-presenting cells (e.g., B cells) d) blocking the activation of B cells e) cytokines from antigen-presenting cells, which may or may not induce depletion; f) tumor cell apoptosis, which may or may not inhibit or reduce tumor cell apoptosis; g) inhibiting tumor cell proliferation; h) may or may not be tumor cell killing agents; i) may or may not stimulate an anti-tumor T cell response; and / or j) may or may not shrink established tumors. The described antibodies may possess or induce any one or more combinations of these attributes or activities. Tai, et al., Cancer Res. 2005, 1;6 5(13):5898-906;Luqman et al.,Blood 112:7 11-720, 2008. The antibodies or portions thereof described herein may also be used to inhibit CD40 inhibition. Tested for effects on ternalization, in vitro and in vivo efficacy, etc. Such assays can be performed using well-established protocols known to those skilled in the art (e.g., Current Protocols in Molecular Biology(G reene Publ.Assoc.Inc.& John Wiley & Sons ,Inc.,NY,NY);Current Protocols in Immu nology(Edited by:John E.Coligan,Ada M.Kr uisbeek, David H. Margulies, Ethan M. Shevac h,Warren Strober 2001 John Wiley & Sons, NY, NY); or can be performed using commercially available kits.

[0142] Condition to be treated The antibodies of the invention, or antigen-binding portions thereof, can be used in vitro and in vivo for therapeutic, prophylactic and therapeutic purposes. and / or have diagnostic utility. For example, cells may be cultured in vitro in culture medium and the cells may be cultured with anti-C The antibody or antigen-binding portion thereof can be contacted with a D40 antibody or fragment thereof. administered as part of an in vivo (e.g., therapeutic or prophylactic) protocol in the body. In an in vivo embodiment, the contacting step occurs within a subject and involves administering an anti-CD40 antibody or a portion thereof in a subject, and in said subject, said antibody or a portion thereof binding to CD40. The antibody or antigen-binding portion thereof is administered under conditions effective to allow , reducing the likelihood of transplant rejection or prolonging the time to transplant rejection, immunosuppression The compounds may be administered to induce apoptosis or to treat an autoimmune disorder.

[0143] The antibodies or antibody fragments described herein can be used in any setting where immunosuppression is desired (e.g., transplantation). Such antibodies may be used in the treatment of transplant rejection or autoimmune disorders. Treatment of the reaction, e.g., reducing the likelihood that the host will reject a particular transplant or causing rejection The antibodies or antibody fragments described herein are particularly useful in prolonging the time to transplantation. Non-limiting exemplary organs and tissues can be used in conjunction with transplantation of any organ or tissue. non-limiting exemplary tissues include heart, kidney, lung, liver, pancreas, intestine, and thymus; Examples include bones, tendons, corneas, skin, heart valves, veins, and bone marrow. The body fragments may also be used to treat autoimmune disorders. Disorders can be associated with or caused by the presence of autoantibodies. Autoimmune diseases that can be treated with the antibody or fragment thereof of the present invention include, but are not limited to, However, systemic lupus erythematosus (SLE), CREST syndrome (calcinosis, Raynaud's syndrome) syndrome, esophageal dysmotility, sclerodactyly and telangiectasia), opsoclonus, inflammatory myocardium myositis (e.g., polymyositis, dermatomyositis, and inclusion body myositis), systemic sclerosis, primary biliary tract infections Liver cirrhosis, celiac disease (e.g., gluten-sensitive enteropathy), dermatitis herpetiformis, Miller-Finnish Sher syndrome, acute motor axonal neuropathy (AMAN), multi- neuropathy with conduction block Focal motor neuropathy, autoimmune hepatitis, antiphospholipid syndrome, Wegener's granulomatosis , microscopic polyangiitis, Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmune hepatitis inflammation, scleromyositis, myasthenia gravis, Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Reb's disease, paraneoplastic cerebellar degeneration, stiff-person syndrome, limbic encephalitis, Isaac syndrome Sydenham's syndrome, Sydenham's chorea, Pediatric autoimmune neuropsychiatric disorders associated with streptococcal amyloidosis (PANDAS) Other autoimmune disorders include encephalitis, type 1 diabetes mellitus, and neuromyelitis optica. , pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease, psoriatic arthritis, Sjögren's syndrome, red lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus); These include multiple sclerosis and reactive arthritis.

[0144] Additional disorders that can be treated using the methods of the present disclosure include, for example, polymyositis, skin Myositis, polyendocrine deficiency, Schmidt syndrome, autoimmune uveitis, adrenal inflammation, thyroid inflammation, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis , presenile dementia, demyelinating disease, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris , pemphigus, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, adult-onset diabetes mellitus (e.g., type II diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcers colitis, Crohn's disease, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, Year-onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasti Chagas' syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid Antibody syndrome, farmer's lung, erythema multiforme, post-open heart syndrome, Cushing's syndrome, autoimmune chronic Active hepatitis, bird enthusiast's disease, allergic disease, allergic encephalomyelitis, toxic epidermal necrolysis, prolapse Alveolitis, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease , erythema nodosum, pyoderma gangrenosum, transfusion reactions, leprosy, malaria, leishmaniasis, Trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell carcinoma Alveolar arteritis, ascariasis, aspergillosis, Sumpter's syndrome, eczema, lymphomatoid granulomatosis , Behçet's disease, Kaplan's syndrome, Kawasaki disease, dengue fever, endocarditis, endomyocardial fibrosis, Endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Fel Tee's syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuchs' cyclitis inflammation, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, Human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus rubella infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Hodgkin's and non- Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, recurrent multiple myeloma chondritis, malignant melanoma, cryoglobulinemia, Waldenström's macroglobulinemia Epstein-Barr virus infection, mumps, Evans syndrome, and autoimmune diseases Examples include epidemiological glandular dysfunction.

[0145] In another embodiment, the antibodies or fragments thereof of the present invention are directed to various antigens associated with the expression of CD40. The compounds may be used in the treatment of disorders such as:

[0146] The disorder can be any condition that can benefit from treatment with an antibody or fragment thereof of the invention. This includes chronic and acute disorders or diseases that predispose a mammal to the disorder. Pathological conditions are also encompassed. Non-limiting examples of disorders to be treated herein include: Autoimmune diseases, immunological disorders, inflammatory disorders, cancer, hematological malignancies, benign and malignant tumors, white These include blood diseases, malignancies of the lymphatic system, and disorders of the vascular system.

[0147] As used herein, the term "CD40-associated disorder" or "CD40-associated disease" refers to a disorder characterized by a CD40-associated 40 expressing cells are characterized by abnormal proliferation or loss of cells. CD40-expressing cells that are associated with cancerous or malignant growth CD40-associated disorders include, but are not limited to, diseases and disorders of the immune system, e.g. For example, autoimmune and inflammatory disorders are included. However, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren's disease syndrome, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), These include lung inflammation, asthma, and idiopathic thrombocytopenic purpura (ITP). More specific examples of cancers that exhibit abnormal expression include B lymphoblastoid cells, Burkitt lymphoma, and tumors, multiple myeloma, T-cell lymphoma, Kaposi's sarcoma, osteosarcoma, epithelial and endothelial tumors, pancreatic These include cancers of the lung, breast, ovary, colon, prostate, head and neck, skin (melanoma), bladder, and kidney. Such disorders also include, but are not limited to, leukemia, lymphoma, e.g., B-cell lymphoma, lymphoma and non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia solid tumors, e.g., sarcomas, e.g., osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma, e.g., Examples include ovarian adenocarcinoma, Kaposi's sarcoma / tumor, and squamous cell carcinoma. No. 090,696.

[0148] Furthermore, CD40-expressing cancers that can be treated or prevented by the antibody or fragment thereof of the present invention include is, for example, leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (e.g. Myeloblastic, promyelocytic, myelomonocytic, monocytic or erythroleukemia), chronic leukemia, Chronic myeloid (granulocytic) leukemia or chronic lymphocytic leukemia; polycythemia vera; lymphoma tumors (e.g., Hodgkin's disease or non-Hodgkin's disease); multiple myeloma, Waldenstrom's disease, macroglobulinemia; heavy chain disease; solid tumors, sarcomas and cancers (e.g., fibrosarcoma, myxoma) Efferent sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma dotheliosarcoma, lymphangiosarcoma, lymphangiosarcoma gioendotheliosarcoma), synovium, mesothelioma, Ewing's tumor, smooth Myosinoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma Cancer, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, trachea tributary carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, Cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, neurological cancer Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, Neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma or esophageal Cancer) are also included.

[0149] Also, B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, arthritis) rheumatoid arthritis and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, Psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wege Th2-lymphocytes (e.g., atopic dermatitis, tuberculosis, or graft-versus-host disease) or Th2-lymphocytes (e.g., atopic dermatitis, dermatitis, systemic lupus erythematosus, atopic asthma, rhinitis and conjunctivitis, allergic rhinitis Also provided are methods for treating disorders such as rheumatoid arthritis, ... Included.

[0150] In some embodiments, the immunological disorder is a T cell-mediated immunological disorder, e.g., It is a T-cell cytotoxicity in which the activated T cells involved express CD40. Alternatively, agents may be administered to deplete such CD40-expressing activated T cells. In one embodiment, CD40-expressing activated T cells are depleted by administration of an anti-CD40 antibody or agent. Although resting T cells may be depleted, they are not substantially depleted by anti-CD40 or other drugs. In the above, "not substantially depleted" means that less than about 60% or less than about 70% of resting T cells are depleted. By "depletion" we mean less than or about 80%.

[0151] Combination therapy The antibodies of the invention, or antigen-binding portions thereof, may be administered alone or in combination with one or more other therapeutic agents, e.g. In some embodiments, the anti-CD40 Pharmaceutical compositions comprising an antibody or a fragment thereof may further comprise a compound conjugated to the antibody or a fragment thereof. and a second therapeutic agent, either conjugated or unconjugated. In one embodiment, the second agent may be another monoclonal or polyclonal antibody. In another embodiment, the second agent is an immunosuppressant. In a third embodiment, the second agent is a cytotoxic or cytostatic agent. In four embodiments, the second agent is an activated lymphocyte, a dendritic cell, or a CD40-expressing cancer cell. It may be capable of targeting a receptor or receptor complex other than CD40 on the surface of the subject.

[0152] Such combination therapy may be administered in combination with other treatments, such as a combination therapy, to treat a variety of conditions, including disease parameters (e.g., symptom severity, number of symptoms, or frequency of recurrences). may have an additive or synergistic effect on

[0153] The anti-CD40 antibodies or fragments thereof of the present invention may be administered concurrently with a second therapeutic agent. In certain embodiments, the second therapeutic agent is administered prior to or after administration of the anti-CD40 antibody or fragment thereof. is administered later.

[0154] The antibodies and antibody fragments described herein may be formulated or administered in combination with immunosuppressive drugs. Examples of immunosuppressants include, but are not limited to, calcineurin inhibitors (e.g., cyclosporine Cyclosporine A (Sandimmune®), cyclosporine G, tacrolimus (Prograf®, Protopic®)), mTor inhibitors ( For example, sirolimus (Rapamune®, Neoral®), Musirolimus (Torisel®), zotarolimus and everolimus (Cer tican®), fingolimod (Gilenya®), myriociguat alemtuzumab (Campath®, MabCampath®) , Campath-1H®), rituximab (Rituxan®, MabThera®), anti-CD4 monoclonal antibodies (e.g., HuMax- CD4), anti-LFA1 monoclonal antibody (e.g., CD11a), anti-LFA3 monoclonal antibody monoclonal antibodies, anti-CD45 antibodies (e.g., anti-CD45RB antibodies), anti-CD19 antibodies (e.g., , U.S. Patent Publication No. 2006 / 0280738), monabatacept (Orenc ia®), belatacept, indolyl-ASC (tacrolimus and ascomycin 32-indole ether derivative of benzophenone), azathioprine (Azasan® , Imuran®), lymphocyte immunoglobulin and antithymocyte globulin [U (Atgam®), mycophenolate mofetil (Cellcept®), Mycophenolate sodium (Myfortic®), Dacriz Mab (Zenapax®), basiliximab (Simulect®) ), cyclophosphamide (Endoxan®, Cytoxan®, Neosar(TM), Procytox(TM), Revimmune(TM), Pro Rednisone, prednisolone, leflunomide (Arava®), FK778, FK779, 15-deoxyspergualin (DSG), busulfan (Myleran (registered trademark), Busulfex (registered trademark), fludarabine (Fludara (trademark), methotrexate (Rheumatrex®, Trexall®) (Trademark), etanercept (Enbrel®), adalimumab (Humir®), a (registered trademark), 6-mercaptopurine (Purinethol (registered trademark)), 15 -Deoxyspergualin (Gusperimus), LF15-0195, Bredini , brequinar, and muromonab-CD3 (Orthoclone®) Examples include:

[0155] Methods for assessing the immunosuppressive activity of drugs are known in the art. The length of in vivo survival of transplanted organs with and without physical intervention is related to the suppression of immune responses. It is also used as a quantitative measure of in vitro assays, such as the mixed lymphocyte reaction. (MLR) assay (e.g., Fathman et al., J. Immunol. 1 18:1232-8, 1977); CD3 assay (anti-CD3 antibody (e.g., OKT 3) specific activation of immune cells (e.g., Khanna et al., Tran splation 67:882-9,1999;Khanna et al. 1999) Transplantation 67:S58); and IL-2R uptake Specific activation of immune cells by exogenously added cytokine IL-2 (e.g. Farrar et al., J. Immunol. 126:1120-5,198 1) can also be used.

[0156] Cyclosporin A (CsA; CAS number 59865-13-3; U.S. Patent No. 3,737 ,433) and its analogs can be used as immunosuppressants. Several other cyclosporins and their derivatives and analogs are also known. Phosphorus and its preparations are described, for example, in the 2004 Physicians' Desk Reference ence (registered trademark) (2003) Thomson Healthcare, 58th ed. and U.S. Patent Nos. 5,766,629; 5,827,822; 4,2 No. 20,641; No. 4,639,434; No. 4,289,851; No. 4,38 No. 4,996; No. 5,047,396; No. 4,388,307; No. 4,970 ,076;No. 4,990,337;No. 4,822,618;No. 4,576, 284; 5,120,710; and 4,894,235. .

[0157] Tacrolimus (FK506) has been shown to be effective against leukemia and pediatric leukemia in both its molecular mode of action and its clinical efficacy. It is a macrolide that has roughly the same effect as CsA in terms of l.Today 14:290-5,1993;Schreiber et al.,I Munol.Today,13:136-42,1992); However, such The effective effect is demonstrated at doses 20-100 times lower than CsA (Peters et al. Drugs 46:746-94, 1993). Tacrolimus and its preparations are For example, the 2004 Physicians' Desk Reference® 003) Thomson Healthcare, 58th ed. and U.S. Pat. No. 4,629,494. ,894,366; 4,929,611; and 5,164,495 It has been done.

[0158] Sirolimus (rapamycin) is an antibacterial agent that inhibits the growth of bacteria, such as Streptomyces hygroscopicus (S Immunosuppression that can be produced by Treptomyces hygroscopicus Sirolimus and its analogues are numerous derivatives and Its formulation is known and is described, for example, in 2004 Physicians' Desk Ref. erence® (2003) Thomson Healthcare, 58t h ed., European Patent No. 0467606; International Publication No. 94 / 02136, International Publication No. 94 / No. 09010, No. 92 / 05179, No. 93 / 11130, No. 94 / 0238 Nos. 5, 95 / 14023 and 94 / 02136 and U.S. Pat. No. 5,023 ,262;No.5,120,725;No.5,120,727;No.5,177, No. 203; No. 5,258,389; No. 5,118,677; No. 5,118,6 No. 78; No. 5,100,883; No. 5,151,413; No. 5,120,84 No. 2; and No. 5,256,790.

[0159] In some embodiments, the second agent is a cytotoxic agent, which may be a conventional chemotherapeutic agent. , e.g., doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, Trexate, mitomycin C or etoposide. Also, strong drugs such as , CC-1065 analog, calicheamicin, maytansine, dolastatin 10 analog Rhizoxin, rhizoxin, and palytoxin may be linked to the anti-CD40 antibody or agent.

[0160] In a further embodiment, the second agent is a humanized anti-HER2 monoclonal antibody; UXAN (rituximab; Genentech, Inc., South San Francisco) (Cisco, Calif.); chimeric anti-CD20 monoclonal antibody); OVAREX (AltaRex Corporation,MA);PANOREX(Glaxo W ellcome, NC; Mouse IgG2a antibody; ERBITUX (cetuximab) (I mclone Systems Inc., NY; anti-EGFR IgG chimeric antibody); V ITAXIN(Medlmmune,Inc.,MD);CAMPATH I / H(Le ukosite, MA; humanized IgG1 antibody; Smart MI95 (Protein Design Labs, Inc., CA;Humanized anti-CD33 IgG antibody);Lym phoCide (Immunomedics, Inc., NJ; humanized anti-CD22 Ig G antibody);Smart ID10(Protein Design Labs, Inc. , CA; humanized anti-HLA-DR antibody; Oncolym (Techniclone, In c., CA; radiolabeled mouse anti-HLA-Dr10 antibody; ALLOMUNE (BioT transplant, CA; humanized anti-CD2 mAb; AVASTIN (Genent ech, Inc., CA; anti-VEGF humanized antibody); Epratuzamab (Immu Nomedics, Inc., NJ and Amgen, CA; anti-CD22 antibody); and C EAcide (Immunomedics, NJ; humanized anti-CEA antibody).

[0161] Other suitable antibodies that can be used as the second agent include, but are not limited to, the following antibodies: Original: CA125, CA15-3, CA19-9, L6, Lewis Y, Lewis X, α-Fetop Protein, CA 242, placental alkaline phosphatase, prostate-specific antigen, prostatic acid phosphatase Sphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE- 4, anti-transferrin receptor, p97, MUC1-KLH, CEA, gp100, MA RT1, prostate-specific antigen, IL-2 receptor, CD20, CD52, CD33, CD22, Human chorionic gonadotropin, CD38, mucin, P21, MPG, and Neu oncogene production Examples include antibodies against substances.

[0162] non-therapeutic use The antibodies described herein are useful as affinity purification agents. The fragments are then attached to a solid phase, such as a protein A resin, using methods well known in the art. The immobilized antibody or fragment thereof is then immobilized on the CD40 protein (or The support is then contacted with a sample containing CD40 protein (which is an immobilized fragment thereof). a suitable solvent that removes substantially all material in the sample other than that bound to the antibody Finally, the support is washed with another suitable solution that will release the CD40 protein from the antibody. Wash with solvent.

[0163] The anti-CD40 antibody of the present invention can also be used to detect and / or quantify CD40 protein. diagnostic assays for detecting CD40 expression in, for example, specific cells, tissues, or serum; It is also useful for sase.

[0164] The antibodies described herein can be assayed using any known assay method, such as competitive binding assays, direct binding assays, or immunoassays. They can be used in direct and indirect sandwich assays, as well as immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques,pp.147-158(CRC Press,Inc.198 See 7).

[0165] Pharmaceutical Composition The present disclosure provides an antibody of the present disclosure or a compound thereof formulated together with a pharmaceutically acceptable carrier. Compositions, e.g., pharmaceutical compositions, comprising the antigen-binding moiety(s) are provided. In embodiments, the composition comprises an isolated nucleic acid encoding an antibody of the invention or an antigen-binding portion thereof. The composition may comprise an acid and a pharmaceutically acceptable carrier. Induce immunosuppression to reduce the severity or prolong the duration of transplant rejection The compositions of the present invention may be effective for treating an autoimmune disorder in a subject. The product can be effective in any of the methods described herein.

[0166] Pharmaceutically acceptable carriers include any suitable solvent that is physiologically compatible. solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Depending on the route of administration, the antibody (or antigen-binding portion(s) thereof) of the invention may be administered in the following manner: The antibody (or antigen-binding portion(s) thereof) is / are reacted with an acid and the antibody (or antigen-binding portion(s) thereof (multiple) antigen-binding moieties) from the action of other natural conditions that may inactivate them. The carrier may be, for example, water, ethanol, polyol (e.g., glycerol), glycol, propylene glycol and liquid polyethylene glycol), and appropriate The mixture may contain a solvent or dispersion medium. Which coating is used, maintaining the required particle size in the case of dispersions and the use of surfactants In certain embodiments, the compositions of the present invention may be maintained by the use of an isotonic agent, e.g. For example, sugars, polyhydric alcohols (mannitol, sorbitol, etc.) or sodium chloride may be added to the composition. The sustained absorption of the injectable composition can be achieved by incorporating an active ingredient into the composition. This can be achieved by including agents that enhance the viscosity, such as monostearate and gelatin. do.

[0167] The pharmaceutical compositions of the invention comprise an antibody or fragment thereof of the invention and a second therapeutic agent described herein. The therapeutic agent may include an agent (eg, one or more immunosuppressant drugs).

[0168] The composition may be in the form of a solution, suspension, emulsion, infusion device, or implantable delivery device. or in solid form ( ) to be reconstituted with water or another suitable vehicle immediately before use. The composition may be presented as an oil emulsion, a water-in-oil emulsion, an oil-in-water emulsion, a dry powder, or the like. Water-in-water emulsion, site-specific emulsion, long-lasting emulsion, viscous emulsion, microemulsion, emulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as non-absorbable impermeable polymers, e.g., ethylenediamine ethylene vinyl acetate copolymers and Hytrel® copolymers, swellable polymers; For example, hydrogels, or absorbable polymers such as collagen and certain polyacids or polyesters. in the form of a steril (such as those used to make absorbable sutures) The compound may be in a form capable of sustained release.

[0169] The compositions may be in the form of pills, tablets, capsules, liquids or sustained release formulations for oral administration. tablets; or liquids for intravenous, intrathecal, subcutaneous or parenteral administration; or for topical administration The sustained release vehicle may be in the form of a polymer or other sustained release vehicle.

[0170] In one embodiment, if the composition is water soluble, the solution of the composition is dissolved in a pharmaceutically acceptable carrier. The compound is dissolved in an aqueous carrier, such as water, saline, or phosphate buffered saline. Acid-buffered saline, Hank's solution, Ringer's solution, dextrose / saline, glucose If necessary, the formulation may contain pharmaceutically acceptable solutions to approximate physiological conditions. It may contain auxiliary substances such as buffers, tonicity adjusters, wetting agents, detergents, etc. The additives may also include further active ingredients, such as bactericides or stabilizers. For example, the solution may contain sodium acetate, sodium lactate, sodium chloride, Potassium, calcium chloride, sorbitan monolaurate or triethanolamine oleate Solid formulations may also be used in the present disclosure. The compositions may be formulated, for example, as pills, tablets, powders, or capsules. Conventional solid carriers can be used, such as mannitol, lactose, dextran, etc. Starch, magnesium stearate, sodium saccharin, talcum, cellulose, Suitable pharmaceutical excipients include glucose, sucrose, magnesium carbonate, etc. Examples of suitable sugars include starch, cellulose, talc, glucose, lactose, sucrose, and zeolite. Latin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, stearic acid Sodium Phosphate, Glycerol Monostearate, Sodium Chloride, Skim Milk Powder, Glycerol Examples of suitable solvents include alcohol, propylene glycol, water, and ethanol.

[0171] Methods well known in the art for making formulations are described, for example, in "Remingt on:The Science and Practice of Pharmacy” (20th ed., ed. AR Gennaro AR., 2000, Lippin Scott Williams & Wilkins, Philadelphia, PA) It is a good idea to take a look at this.

[0172] In one aspect, a pharmaceutical formulation comprising a composition or a nucleic acid, polypeptide or antibody of the invention is Incorporated into a lipid monolayer or bilayer, e.g., a liposome. U.S. Patent No. 6,110,4 No. 90; No. 6,096,716; No. 5,283,185 and No. 5,279,8 No. 33. Also, in accordance with various aspects of the present invention, the water-soluble nucleic acid, peptide or polypeptide of the present invention The present invention provides a formulation in which a peptide is attached to the surface of the monolayer or bilayer. Razide-PEG-(distearoylphosphatidyl)ethanolamine-containing liposomes ( See, for example, Zalipsky, Bioconjug. Chem. 6:705-708, 19 95). Liposomes or any form of lipid membrane, e.g., planar lipid membranes or Alternatively, intact cells, such as red blood cell membranes, may be used. Liposomal formulations may be used in any means, for example, intravenous administration, transdermal administration (see, e.g., Vutla, J. Pharm. Sci. 8 5:5-8, 1996), transmucosal administration, or oral administration. According to the invention, the nucleic acids, peptides and / or polypeptides of the invention can be incorporated into micelles and / or liposomes. and providing pharmaceutical preparations incorporated within the formulation (see, e.g., Suntres, J. Pharm. .Pharmacol.46:23-28,1994;Woodle,Pharm.Re (See s.9:260-265, 1992). Liposomes and liposome formulations are prepared using standard methods. These compounds can be prepared according to methods well known in the art. tokines Mol. Ther. 1:197-210, 1995. Alving,I mmunol.Rev.145:5-31,1995. Szoka,Ann.Rev.B Biophys.Bioeng.9:467,1980. U.S. Patent No. 4,235,871 ;Ibid. Nos. 4,501,728 and 4,837,028.

[0173] In one embodiment, the composition comprises a carrier that protects the peptide from rapid elimination from the body, e.g. For example, controlled release formulations, such as those prepared using implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycols, Colic acid, collagen, polyorthoesters and polylactic acid may also be used. Those skilled in the art will know how to prepare such formulations. It may also be used as a carrier for obtaining the desired compound. U.S. Patent No. 4,522,811.

[0174] The compositions of the present disclosure can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Administration can be parenteral, intravenous, intrathecal, subcutaneous, oral, topical, local, intramuscular, intradermal, transdermal, or transdermal. Intravenous delivery by continuous infusion can be intravenous, intrarectal, spinal, or transepithelial. 1 is an example of an exemplary method for administration.

[0175] In order to administer the drug of the present invention by a specific administration route, the drug is administered by a method that inhibits its inactivation. It may be necessary to coat the agent with or co-administer the agent with a material that For example, the agent can be administered to a subject in an appropriate carrier, for example, in liposomes, or in a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes. Examples include liposomes (Strejan et al., J. Neuroimmunol .7:27-41,1984).

[0176] Parenteral administration includes modes of administration other than enteral and topical administration, usually by injection. The administration of the drug may be achieved by any of the following means, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, Intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, epidural and sternal Suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include intravenous injection and infusion. Examples of aqueous and non-aqueous carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, Examples include olive oil and injectable organic esters such as ethyl oleate. Proper fluidity can be achieved, for example, by the use of coating materials, such as lecithin, in the case of dispersions This can be achieved by maintaining the required particle size and by using surfactants.

[0177] Methods for preparing parenterally administrable compositions are known or will be apparent to those skilled in the art. The details are reported in Bai, J. Neuroimmunol. 80:65-7. 5,1997. Warren, J. Neurol. Sci. 152:31-38,199 7. Tonegawa, J. Exp. Med. 186:507-515, 1997. Formulations for oral administration may contain, for example, excipients, sterile water, saline, polyalkylene glycol, The oils may contain alcohols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible and biodegradable lactide polymers, lactide / glycolide copolymers, or The polyoxyethylene-polyoxypropylene copolymers are useful for controlling the release of the drugs of the present invention. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles) can be used to , liposomes) can be used to control the biodistribution of the agents of the present invention. Useful delivery systems for this include ethylene-vinyl acetate copolymer particles, osmotic pumps, and intrathecal These include pumps, implantable infusion systems, and liposomes. will vary depending on several factors, such as the dosage of the drug administered and the route of administration.

[0178] Sterile injectable solutions can be prepared by incorporating the agent of the present invention in the required amount in an appropriate solvent, as required. Incorporated with one or a combination of the ingredients listed above, followed by sterile microfiltration Generally, the dispersant can be prepared by dissolving the agent of the present invention in a base dispersant. in a sterile vehicle containing the solvent and any other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation The method involves the step of preparing powders of the active ingredient plus any additional desired ingredients from these previously sterile filtered solutions. The dosage regimen includes vacuum drying and freeze drying (lyophilization) which are used for the preparation of pharmaceuticals. The dosage is adjusted to provide the optimum desired response (e.g., a therapeutic response). A bolus may be administered or several divided doses may be administered over time, depending on the requirements of the therapeutic situation. For example, the antibody of the invention may be administered in a dose of: It may be administered by subcutaneous injection once or twice a week, or once or twice a month by subcutaneous injection. Parenteral compositions may be administered in unit dosage form for ease of administration and uniform dosage. A unit dosage form, as used herein, refers to a single dose administered to a subject to be treated. Each unit is a physically discrete unit suitable as a dosage unit for achieving a desired therapeutic effect; A predetermined amount of active agent calculated to provide the desired effect is mixed with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are as follows: (a) the specific characteristics of the active agent and (b) the specific therapeutic effect sought to be achieved, and (b) such active agent for treating the hypersensitivity of the individual. It is determined by and directly depends on the limitations inherent in the technical field of drug formulation.

[0179] When administered orally, the compositions of the invention may be protected from digestion. To make the antigen-binding portion resistant to acid hydrolysis and enzymatic hydrolysis or conjugating the antibody or antigen-binding portion thereof to a suitably resistant carrier, This can be done by either encapsulating the drug in a liposome, for example. Protective measures are well known in the art. Fix, Pharm Res. 13:1 760-1764,1996. Samanen, J.Pharm.Pharmacol. 48:119-135, 1996. U.S. Patent No. 5,391,377.

[0180] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and can be used, for example, for transmucosal administration. Examples of permeability enhancing agents include bile salts and fusidic acid derivatives. Detergents also aid in permeation. Transmucosal administration can be through the use of nasal sprays or suppositories. Sayani, Crit. Rev. Ther. Drug Carrier Sy st.13:85-184,1996. For topical or transdermal administration, the drug is applied as an ointment. They are formulated into creams, salves, powders and gels. Transdermal delivery systems include, for example, For example, patches may be mentioned.

[0181] The compositions of the present invention may also be administered by sustained delivery or slow release mechanisms. For example, biodegradable Microspheres or capsules or other biodegradable polymers capable of sustained delivery of peptides mer constructs can be included in the formulations of the present invention (see, e.g., Putney, Nat. Biotech. hnol.16:153-157,1998).

[0182] For inhalation, the compositions of the present invention can be administered in any system known in the art, e.g. , dry powder aerosols, liquid delivery systems, air-jet nebulizers, propellant systems, etc. Patton, Biotechniques 16:141-143, 1 998. Also of use in the present disclosure are, for example, Dura Pharmace uticals(San Diego,Calif.), Aradigrn(Haywa rd,Calif.), Aerogen(Santa Clara,Calif.), I nhale Therapeutic Systems(San Carlos,Cal) if.) and other products and inhalation delivery systems for polypeptide macromolecules. The formulation may be administered in the form of an aerosol or mist. For aerosol administration, the formulation The formulation may be supplied in finely divided form along with a surfactant and propellant. The device for delivering the agent to the respiratory tissues is an inhaler, which vaporizes the formulation. Drug delivery systems include, for example, air-jet nebulizers.

[0183] The composition may be administered in a single dose treatment, or in multiple dose treatments depending on the age, weight and Administered on a schedule and duration appropriate to the patient's physical condition, the particular composition used, and the route of administration. The frequency of administration can be determined by any of a variety of factors, including the severity of symptoms, the desired immunity, and the Depending on the degree of protection provided and whether the composition is used for prophylactic or curative purposes, For example, in one embodiment, the compositions according to the present invention may be administered once a month, twice a month, or three times a month. times, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, 5 times a week, 6 times a week, every other day (qod), daily (qd), twice a day (qid) or three times a day (tid) is administered.

[0184] The duration of administration of the polypeptides according to the present invention, e.g., the period during which the composition is administered, can be any The amount of the composition may vary depending on a variety of factors, such as the response of the subject. About 1 day to about 1 week, about 2 weeks to about 4 weeks, about 1 month to about 2 months, about 2 months to about 4 months Months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 Administered for a period ranging from one year to approximately two years, or from approximately two years to approximately four years, or longer It can be done.

[0185] Oral or parenteral compositions are prepared in unit dosage forms for ease of administration and uniform dosage. Unit dosage form, as used herein, refers to a dosage form that is conveniently formulated to provide a single dose to the patient to be treated. A physically discrete unit suitable as a dosage unit for a subject; each unit may contain the desired A predetermined amount of active agent calculated to produce a therapeutic effect is administered to the patient in the required pharmaceutical carrier. It is contained in an associated state with the body.

[0186] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure will vary depending on the particular patient, composition and dosage regimen. an activity effective in a manner of administration to produce the desired therapeutic response without being toxic to the patient; The dosage levels selected may be varied to achieve various pharmacokinetic The specific composition of the present disclosure or its esters, salts or analogs used may be used. activity of the amide, route of administration, duration of administration, rate of excretion of the specific drug used, duration of treatment , other drugs, agents and / or substances, treatments used in combination with the specific composition used The patient's age, sex, weight, physical condition, general health condition and medical history are all well known in the medical field. A physician or veterinarian of ordinary skill in the art will be able to determine the required The effective amount of the pharmaceutical composition to be administered can be readily determined and prescribed by, for example, a physician or veterinarian. Therefore, the dose of the agent of the present invention used in the pharmaceutical composition is determined based on the amount of the agent required to achieve the desired therapeutic effect. The dosage is started at a lower level than that prescribed and gradually increased until the desired effect is achieved. Generally, a suitable daily dose of the composition of the present invention is an amount effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the therapeutic composition may be administered in separate (unit dose) doses at appropriate intervals throughout the day. as 2, 3, 4, 5, 6 or more divided doses administered in separate doses (which may be in pharmaceutical form). You may do so.

[0187] Data obtained from cell culture assays and animal studies may be used to determine the dosage for human use. In one embodiment, the dosage of such agents is such that there is little to no toxicity. Little or no ED 50 The dosage is within a range of circulating concentrations that includes The ranges may vary depending on the dosage form used and the route of administration used. In certain conditions, the therapeutically effective dose can be estimated initially from cell culture assays. I C 50 (i.e., the concentration of test drug that results in half-maximal inhibition of symptoms) Doses that achieve a plasma concentration range can be established in animal models. ,Springer,Sem.Immunopathol.25:35-45,2003 . Nikula et al.,Inhal.Toxicol.4(12):123-5 3,2000.

[0188] Exemplary, Non-Limiting Ranges for Therapeutically or Prophylactically Effective Amounts of Antibodies or Antigen-Binding Portions of the Invention Approximately 0.001 to approximately 100 mg / kg body weight or more, approximately 0.1 to approximately 100 mg / kg body weight, about 0.01 to about 80 mg / kg body weight, about 0.001 to about 60 mg / kg body weight, About 0.01~about 30mg / kg body weight, about 0.01~about 25mg / kg body weight, about 0.5~about 25mg / kg body weight, about 0.1 to about 15mg / kg body weight, about 0.1 to about 20mg / kg body weight Weight, about 10 to about 20 mg / kg body weight, about 0.75 to about 10 mg / kg body weight, about 1 to about 10 mg / kg body weight, about 2 to about 9 mg / kg body weight, about 1 to about 2 mg / kg body weight, about 3 to about 8 mg g / kg body weight, about 4 to about 7 mg / kg body weight, about 5 to about 6 mg / kg body weight, about 8 to about 13 m g / kg body weight, about 8.3 to about 12.5 mg / kg body weight, about 4 to about 6 mg / kg body weight, about 4 .2~about 6.3mg / kg body weight, about 1.6~about 2.5mg / kg body weight, about 2~about 3mg / kg body weight, or about 10 mg / kg body weight. The dosage administered to the subject is about 0. 1mg / kg ~ approx. 50mg / kg, approx. 1mg / kg ~ approx. 30mg / kg, approx. 1mg / kg about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg body weight of the subject. Exemplary doses include, but are not limited to, 1 ng / kg In some embodiments, the dose is about 0.5 mg / kg or less. , about 1mg / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg , about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about 10mg / kg g, about 11mg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 1 5 mg / kg or about 16 mg / kg body weight of the subject. WO 94 / 04188.

[0189] The composition is formulated to contain an effective amount of an antibody of the invention or an antigen-binding portion thereof. wherein the amount depends on the animal being treated and the condition being treated. The antibody or antigen-binding portion thereof of the invention may be used in an amount of about 0.01 mg to about 10 g, about 0.1 mg to about 9 g , about 1mg to about 8g, about 1mg to about 7g, about 5mg to about 6g, about 10mg to about 5g, about 2 0mg to about 1g, about 50mg to about 800mg, about 100mg to about 500mg, about 0.01 mg to about 10 g, about 0.05 μg to about 1.5 mg, about 10 μg to about 1 mg of protein, Approximately 30μg to approximately 500μg, approximately 40pg to approximately 300pg, approximately 0.1μg to approximately 200mg, Approximately 0.1μg to approximately 5μg, approximately 5μg to approximately 10μg, approximately 10μg to approximately 25μg, approximately 25μg ~Approx. 50μg, approx. 50μg ~ approx. 100μg, approx. 100μg ~ approx. 500μg, approx. 500μg The dose ranges from about 1 mg to about 1 mg, and from about 1 mg to about 2 mg for any particular subject. The specific dose level to be administered will depend on various factors, e.g., the activity of the specific peptide, age, Body weight, general health condition, sex, dietary habits, administration period, administration route and excretion rate, concomitant use of drugs, and It depends on the severity of the particular disease being treated.

[0190] manufactured goods In another aspect, a composition comprising substances useful for treating a condition or disorder described herein is provided. The article of manufacture includes a container and a label. Suitable containers include: Examples include bottles, vials, syringes, and test tubes. Containers can be made of a variety of materials. The container may be formed of, for example, glass or plastic. The container may hold an active composition and have a sterile access port. For example, May be an intravenous solution bag or a vial having a stopper and pierceable by a hypodermic needle The active agent in the composition is a humanized anti-CD40 antibody or fragment thereof, or a humanized anti-CD40 antibody or fragment thereof described herein. It may be any other antibody or fragment thereof. The label on or associated with the container may include: The composition is indicated to be used to treat the condition of choice. The article of manufacture may further comprise a pharmaceutically acceptable buffer, such as phosphate buffered saline, A second container containing Ringer's solution and dextrose solution may also be included. , and other substances that are commercially available and desirable from a user's perspective, e.g., other buffers. , diluent, filter, needle, syringe and package insert with instructions for use It may also be something.

[0191] In one embodiment, the present invention provides a method for the treatment of rhesus mastitis comprising administering to a subject an anti-CD40 antibody or antigen-binding portion thereof. Further components of the kit include: instructions for use; other reagents, treatments, etc. a therapeutic agent, or an agent useful for coupling an antibody to a label or therapeutic agent, or Other materials for preparing the antibody for administration; a pharmaceutically acceptable carrier; and the antibody to be administered to a subject. The present invention may include one or more of the following: a device or other substance for administering the treatment;

[0192] The kit may or may not include a second therapeutic agent as described herein. The agents may be mixed together in a kit or may be individually packaged. good.

[0193] The kit comprises at least one nucleic acid encoding an anti-CD40 antibody or fragment thereof; It may or may not include instructions for expression of the nucleic acid. Other possible components of the kit include expression vectors and cells.

[0194] Use of the antibodies of the present invention or fragments thereof in diagnostic kits, i.e., for performing diagnostic assays. It may be used in combination with predetermined amounts of reagents packaged together with instructions. If the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme, For example, a substrate precursor may be included that provides a detectable chromophore or fluorophore. Also, other additives, such as stabilizers, buffers (e.g., block buffers or lysis buffers), The concentration in the reagent solution at which the sensitivity of the assay is substantially optimized may be included. The relative amounts of the various reagents to provide the desired concentration can vary widely. It is provided as a dry powder (usually lyophilized) containing excipients that result in a reagent solution with a high viscosity. It can be done.

[0195] Epitope mapping Methods for identifying the specific epitope to which an antibody binds are known to those skilled in the art. Standard techniques include peptide scanning, which identifies peptides to which antibodies bind. Short overlapping peptides (e.g., 10–30, e.g., 10–30) derived from the full-length protein of the elephant For example, 20 amino acids in length are individually tested for their binding ability to the antibody. Such experiments can then determine the region of the protein to which the antibody binds. .

[0196] Site-directed mutagenesis can also be used to identify the antigenic region(s) of a particular protein. In this approach, point mutations are systematically introduced into a target polypeptide. The ability of antibodies to bind to peptides containing mutations at various positions is used to identify the protein. It is then determined whether a particular region of the substrate contains the epitope to which the antibody binds.

[0197] Alternatively, antibody epitopes can be identified using high-throughput mutagenesis techniques, e.g., shotgun mutagenesis. Induction (Shotgun Mutagenesis) (Integral Molecul ar, Inc., Philadelphia, Pa.) (This is because The method can also be used to generate quantitative variations. The method allows for efficient epitope identification within proteins.

[0198] To investigate whether various antibodies against CD40 bind to similar epitopes, In vitro competitive blocking assays can be performed. In one embodiment, chimeric IgG1 CD40-specific antibodies 2C10, 3A8, and Chi220 were used in this assay. 2C10 was used for allophycocyanin (APC) and Lightning Link antibody. Using a nucleotide labeling kit (Novus Biologics, Littleton, CO) Human PBMCs were conjugated with increasing concentrations of 2C10, 3A8, or Chi220. The antibodies were incubated together and then stained with the APT conjugate 2C10. The ability of APC-conjugated 2C10 to cross-block 1C10 was assessed. As shown in Figure 2, the concentration of 2C10 decreased with increasing concentration, but the concentration of Chi220 or 3 This result indicates that 2C10 is more efficient than either Chi220 or 3A8. It is shown that the antibody binds to a unique epitope that is distinct from both the IgG and IgG antibodies.

[0199] CD40 fragment The present invention also provides a CD40 antibody containing the epitope specifically bound by the 2C10 antibody. The 2C10 antibody is directed against the extracellular portion of the CD40 polypeptide. The 2C10 antibody reacts with a portion of this sequence (SEQ ID NOs: 5 and 6). .

[0200] Thus, in the present disclosure, CD40 fragments (e.g., CD40 fragments) that are specifically bound by the 2C10 antibody are For example, less than 150, 120, 100, 80, 70, 60, 50, 40, 30, 2 The amino acid length is specified as follows: 0, 18, 15, 12, 11, 10, 9, 8, or 7. In certain embodiments, the fragments are 8 to 10, 8 to 12, 8 to 15, 8 to 20, 8 ~30, 8~40, 8~50, 8~60, 8~70, 8~80 or 8~100 amino acids In other embodiments, the fragment is 7-10, 7-12, 7-15, 7-20, 7 Lengths of up to 30, 7-40, 7-50, 7-60, 7-70, 7-80 or 7-100 be.

[0201] The 2C10 antibody contains amino acids 8 to 10, 8 to 12, 8 to 15, 8 to 20, and 8~30, 8~40, 8~50, 8~60, 8~70, 8~80, 8~100, 7~10 , 7~12, 7~15, 7~20, 7~30, 7~40, 7~50, 7~60, 7~70 , and binds to an epitope present within 7 to 80 or 7 to 100 sequences.

[0202] The present invention also features fusion proteins comprising the fragments described herein and heterologous sequences. In certain embodiments, one of the fusion partners is an Fc protein (e.g., For example, mouse Fc or human Fc. One of the fusion partners may be a fusion protein useful for antibody production. In other embodiments, the sequence may be a maltose binding protein or GST. Alternatively, the fusion protein may contain a purification or detection tag, e.g., a tag that can be detected directly or indirectly. proteins, such as green fluorescent protein, hemagglutinin, or alkaline phosphatase), DNA binding domains (e.g., GAL4 or LexA), gene activation domains (e.g., GAL4 or VP16), a purification tag, or a secretory signal peptide (e.g., promoter The signal sequence is the preprotyrypsin signal sequence. In some embodiments, the fusion partner may be a tag, such as c-myc, polyhistidine, or FLAG. Each fusion partner may comprise one or more domains, e.g., a preprotrypsin signaling domain. It may contain a null sequence and a FLAG tag.

[0203] The CD40 fragments and fusion proteins described herein can be expressed by inducing suitable host cells to express the polypeptide. A polynucleotide molecule encoding the peptide fragment or fusion protein is inserted into a suitable expression vehicle. It can be produced by transformation with

[0204] Any of a wide variety of expression systems can be used. Exemplary expression systems include prokaryotic hosts (e.g., E. coli) and eukaryotic hosts (e.g., S. cerev) isiae), insect cells, such as Sf21 cells, or mammalian cells, such as NIH 3T 3, HeLa, or preferably COS cells). Such cells are widely available. Former (e.g., American Type Culture Collection, M Transformation or transfection methods The choice of the expression vehicle will depend on the host system selected. The method is described, for example, in Kucherlapati et al. (CRC Crit. R Biochem. 16:349-379, 1982) and DNA Transfer r to Cultured Cells(eds.,Ravid and Fresh ney, Wiley-Liss, 1998); expression vehicles are described, for example, in V sectors:Expression Systems:Essential Tech niques(ed.,Jones,Wiley & Sons Ltd.,1998) The ion exchangeable ...

[0205] Once the recombinant CD40 polypeptide fragment or fusion protein is expressed, it can be used in, for example, They can be isolated using affinity chromatography. In one example, A suitable antibody (e.g., an antibody or fragment thereof described herein) is bound to the column, and the polypeptide is Affinity chromatography can be used to isolate peptide fragments or fusion proteins. Prior to purification, lysis and fractionation of cells bearing the fragment or fusion protein was performed using standard methods. (See, for example, Methods in Enzymology, vol. me 182, eds., Abelson, Simon, and Deutscher, (See Elsevier, 1990). Once isolated, the CD40 polypeptide fragment or fusion The protein may be further purified, if desired, for example by high performance liquid chromatography. (e.g., Fisher, Laboratory Techniques in B iochemistry and molecular biology, eds.,W ork and Burdon, Elsevier, 1980; and Scopes, Pr otein Purification: Principles and Practice ce,Third Edition,ed.,Cantor,Springer,199 See 4).

[0206] CD40 polypeptide fragments or fusion proteins can also be produced by chemical synthesis. (e.g., Solid Phase Peptide Synthesis, 2nd ed.,1984,The Pierce Chemical Co.,Roc kford, IL; and Solid-Phase Synthesis: A Practice ical Guide, ed., Kates and Albericio, Marce. l by the method described in Dekker Inc., 2000).

[0207] The antibodies, antigen-binding portions thereof, compositions and methods of the invention can be used in any vertebrate, including mammals. Mammals and non-mammals, e.g., humans, mice, rats, guinea pigs, hamsters, dogs , cat, cow, horse, goat, sheep, pig, monkey, ape, gorilla, chimpanzee, rabbit It can be used in geese, ducks, geese, chickens, amphibians, reptiles and other animals.

[0208] The following examples of specific modes for carrying out the present disclosure are presented for illustrative purposes. It is merely an example and is not intended to limit the scope of the present disclosure in any way.

[0209] [Example 1] Generation and identification of anti-CD40 mouse antibodies Mice (strain AJ) were transfected with rhesus monkeys (rhes macaque (M. mulatta) CD40 (amino acid sequence: EPPTAC REKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCSESE FLDTWNRETRCHQHKYCDPNLGLRVQQKGTSETDTICTCE EGLHCMSESCESCV; a fusion protein consisting of the extracellular domain of SEQ ID NO: 5) Rhesus macaques were immunized with CD40-MBP. The amino acid sequence of this region of the CD40 protein is identical to that of the human CD40 protein, with five amino acids. The human amino acid sequence differs in the amino acid position: EPPTACREKQYLINSQCCS LCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHHQ HKYCDPNGLRVQQKGTSETD TICTCEEGWHCTSEACES CD40-MBP was administered to mice multiple times in complete Freund's adjuvant (CV; SEQ ID NO: 6). The spleen cells of the immunized mice were administered with Freund's adjuvant and incomplete Freund's adjuvant. The hybrid cells were fused with the mouse myeloma cell line SP2 / 0 and then cultured as standard hybridomas. Technique was used to select.

[0210] The antibody was expressed against the same rhesus CD40 domain fused to glutamine synthetase. The antibodies were selected for their reactivity to a second fusion protein (CD40-GST) consisting of the main fusion protein. Antibodies reactive with CD40-GST (by ELISA) were isolated from rhesus monkeys (rhesus macaques). macaque blood B cells, human blood B cells and rhesus macaque (rhesus macaque) blood B cells Reactivity to native CD40 expressed on caque B lymphoblastoid cell lines As a final level of selection, antibodies were further tested by in vitro assay. Human CD154-expressing Jurkat D1.1 cells after co-culture in vitro assay or their ability to inhibit B cell activation in rhesus macaques. Stable subclones of the anti-CD40 antibody 2C10 were obtained by limiting dilution. The antibody is mouse IgG1-κ.

[0211] Lowe et al.,A novel monoclonal antibody to CD40 prolongs islet allograft surviv al,Am.J.Transplant(2012)12(8):2079-87. Antibody cloning The variable regions of the monoclonal antibody can be cloned using any method known in the art. To obtain antibody variable region sequences for hybridoma cells, PCR-based methods are described, for example, in Larrick et al., Nat. Biotech hnol.7:934-8, 1989 and Orlandi et al., Proc.N atl. Acad. Sci. USA 86:3833-7, 1989. Using this or similar techniques, the variable regions of monoclonal antibodies can be cloned. In the present case, the heavy and light chain variable regions of the 2C10 antibody are The sequence was cloned and sequenced. DNA representing the heavy chain variable region and the light chain variable region was isolated using 5' RACE PCR as follows: DNA primers: Mouse kappa reverse: 5'-CTA ACA CTC ATT CCT GTT GAA GCT CTTGAC (SEQ ID NO: 7); Mouse kappa forward: 5'-GCT GAT GCT GCA CCA ACT GT A TCC-3' (SEQ ID NO: 8) Mouse IgG1 reverse: 5'-GGC AAC GTT GCA GGT CTC GC-3' (SEQ ID NO: 9) Mouse IgG1 forward: 5'-CTG GAT CTG CTG CCC AAA CTA ACT CC-3' (SEQ ID NO: 10) The clone was cloned using

[0212] PCR products were cloned into commercially available cloning vectors and subjected to standard sequencing. The resulting sequence is shown in Figure 1.

[0213] The immunoglobulin variable region genes were cloned into the anti-CD40 antibody clone 2C10 secretory hybrid. Anti-human CD40 clone 3A8 (Kwekkeboom et al., Imm unology 79:439-44,1993)(American Type Cu Culture Collection, ATCC, Vienna, VA) from 5' rapid amplification of cDNA ends DNA ends) - cloned using polymerase chain reaction. The heavy and light chain variable regions of the rhesus IgG1 or rhesus The sequences of the rhesus IgG4 heavy chain and rhesus κ light chain constant regions were The gene was subcloned into an expression vector containing

[0214] The recombinant heavy and light chains were subcloned into expression vectors and expressed in Chinese hamster The retroviral vector used to transduce ovarian cells contains GPEx (trademark ) Expression technology (Catalent Pharma Solutions, Middleton The transduced cell pool was cultured in serum-free medium and packaged using the ELISA kit. The secreted antibody was purified by protein A affinity chromatography. The chimeric rhesus IgG1 (2C10R1, 3A8R1) and IgG4 (2C10R4) antibody was diafiltered in phosphate buffer; Endotoxin levels were determined to be less than 1 endotoxin unit / mg.

[0215] Antibody characterization 2C10 binds to CD40 and prevents CD154 binding The ability of 2C10 to bind to both rhesus and human CD40 was evaluated. To evaluate the efficacy of recombinantly expressed human or rhesus CD40, ELI was used. The antibodies were adsorbed onto SA plates and reacted with various concentrations of 2C10. C10 binding was detected by ELISA using goat anti-mouse IgG-HRP. The results showed that 2C10 binds similarly to rhesus and human CD40. This is important for the clinical translation of 2C10. Confirmation of the ability of 2C10 to block binding of its cognate ligand, CD154 To identify the B cells of rhesus monkeys and humans, we cultured them in increasing concentrations of 2C10 or ibuprofen. Incubation with a type control followed by histidine-tagged soluble CD154 (R and incubated with HCl (Human & D Systems, Minneapolis, MN). 2C10 blocked CD154 binding in a dose-dependent manner. 2C10 binds T cell-associated CD154 and CD40 on B cells and antigen-presenting cells (Figure 3). It has been shown that this can effectively block the interaction with

[0216] Table 2. CD40 receptor binding kinetics of 2C10 [Table 2]

[0217] 2C10 was found to be effective in rhesus monkey and human peripheral blood mononuclear cells. Blocks B cell activation The anti-CD40 antibody 2C10 was tested in rhesus monkeys ( The results were characterized using both rhesus monkey (Rh) and human peripheral blood mononuclear cells (PBMCs). CD20 expression was chosen because it is an indicator of B cells and CD2 3. Because the expression of CD80 and CD86 is associated with B cell activation. The antibodies were first assessed for their ability to bind to CD20. PBMCs were incubated with fluorochrome-conjugated 2C10 and anti-CD20 antibody. Flow cytometry analysis was used to identify the markers in humans and rhesus monkeys. In another set of experiments, we confirmed the binding of 2C10 to CD20+ B cells (Fig. 2A). In the present study, PB derived from either rhesus monkeys or humans was MCs were cultured using the immortalized T lymphocyte cell line, CD154 + Ju The B cells were cultured either in the presence or absence of rkat D1.1 cells. , three markers (CD23, CD8) on CD20+ cells present in PBMCs The general scheme of this assay is shown in Figure 1. As shown in Figure 4, the culture of PBMCs in the presence of Jurkat cells This resulted in increased expression of all three markers, and B cells expressed CD154 + Jurkat Thin It has been shown to be activated by cytoplasmic vesicles.

[0218] To test the ability of the antibodies to block B cell activation, PBMCs and Jurkat cells were cultured. The cells were cultured in the presence and absence of one of three antibodies: 3A8, 5C8, and 2C10. The 3A8 antibody was a mouse anti-human CD40 antibody (ATCC accession number HB-1202 4), and 5C8 is an anti-CD154 antibody (ATCC accession number CRL-10915). Each was used as a positive control. Co-cultures were performed over a five-digit antibody concentration range (0.001 μg to 1 As shown in Figure 5, 3A8 inhibited the activity of rhesus monkeys (rhesus s) Blocks B cell activation as measured by CD23 expression in PBMCs Although the IL-16 receptor agonist did not stimulate activation, both 2C10 and 5C8 were able to block activation with similar efficiency. Corresponding changes were also observed in the expression of CD80 and CD86. The results indicate that 2C10 binds to a different epitope on CD40 than 3A8. These results also suggest that 2C10 acts as a partial agonist with weak stimulatory potential. In contrast to 3A8, which was previously shown to be munol.174:542-50,2005,Badell et al.,Am.J .Transplant.accepted for publication,201 1) and also acts primarily as a CD40 antagonist. When a similar experiment was performed using human PBMCs instead of 2C10, the results were similar to those of 2C10 and 5C8. Both activate B cells with similar efficiency, as measured by CD86 expression. In this case, the 3A8 antibody was observed to block the rhesus macaque Unlike in PBMCs, it blocked B cell activation (Fig. 6).

[0219] The 2C10 and 3A8 antibodies were also tested in a Jerkass study for their ability to activate B cells. In the absence of rat cells, either rhesus monkey or human PBMCs were used. In this case, PBMCs were incubated in the presence of either 2C10 or 3A8. The expression of CD23, CD80, and CD86 was then measured. CD20 + As shown in Figure 7, the rhesus monkey ) CD23 expression on cells was increased in the presence of 3A8 antibody, but not in the presence of 2C10 antibody. In contrast, neither 3A8 nor 2C10 activated human B cells. The difference in activity observed between the 3A8 and 2C10 antibodies is due to the fact that the 2C10 antibody is more potent than the 3A8 antibody. This indicates that the antibody binds to a different epitope than that of the antibody.

[0220] 2C10 interferes with T cell-dependent antibody responses 2C10 binds to a unique epitope on CD40 and inhibits B cell activation by the anti-CD154 antibody. Having established that the compound inhibits ATP-dependently binding to the ATP receptor and lacks agonist properties, the inventors then The effects of 2C10 in vivo were characterized. rhesus) chimeric form, rhesus IgG1 (2C10R1) or IgG4 (2C10R4) heavy chain and rhesus kappa light chain constant The chimeric rhesus Ig of 3A8 was also prepared using the sequence of the common region. The G1 form (3A8R1) was generated for use as a control.

[0221] Rhesus macaques were given 4-hydroxy-3-nitrophenylalanine (4-hydroxy-3-nitrophenylalanine) on day zero. Triphenylacetyl conjugate keyhole limpet hemocyanin (KLH, 10 mg IM ) antigen (Biosearch Technologies, Novato, CA) once. Before and one week after immunization, cohorts of three animals were given an intravenous dose (50 The subjects were administered 2C10R1, 2C10R4, 3A8R1 (mg / kg) or saline. All animals were observed for 70 days and flow cytometry was performed weekly. Treatment with the 2C10 isotype increased the expression of 3A8R1 (Badell et al., Am J. Transplant. 10:214, 2010) or Chi220 (Adams et al., J. Immunol. 174:542-50, 2005) Compared with previously reported significant long-term peripheral B cell depletion occurred in treated animals, Moderate changes in cell counts were produced (Figure 8).

[0222] T cell-dependent antibody responses to KLH-NP were tested by ELISA. coated with KLH (0.01 mg / ml, Sigma, St. Louis, MO). Super Block (Thermo Scientific, Woodstoc The pre- and post-treatment plasma samples were serially diluted and blocked with GA. The cells were then plated and washed with phosphate buffered saline / 0.05% Tween. The body was then incubated with monoclonal anti-rhesus IgG-horseradish peroxidase. Oxidase (clone 1B3, NHP Reagent Resources, Boston Scientific) The plate was then incubated with PBS (MA) for 1 hour. The mixture was incubated with peroxidase substrate solution (KPL) and then with stop solution (KPL ) was added and the optical density was read at 450 nm on an ELISA plate reader. The samples were analyzed to determine whether the optical density readings of the plasma after treatment were greater than the optical density readings of the plasma before treatment at the same 2-fold dilution. A density exceeding that of the control was considered positive at a given dilution. High titers of KLH-specific IgG were generated in the animals treated with 3A8R1 (Fig. 9). Anti-KLH responses were also generated in animals treated with IgG, but titers were significantly higher than those in controls despite significant B cell depletion. In contrast, the production of IgG anti-KLH antibodies was approximately 10-fold lower than that of 2C10R1 or Almost complete blockade was observed by day 56 in all animals receiving either 2C10R4 or 2C10R5. It was clicked.

[0223] 2C10 was shown to be effective in islet allotransplantation in a macaque model of islet allotransplantation. Significantly prolongs graft survival The inventors further discovered a CD4 purified chimeric rhesis IgG4 antibody. 2C10R4 was tested in a non-human primate islet allograft model (FIG. 10). Rhesus macaques weighing 10–20 kg were given a midline injection one day before transplantation. Donor pancreatectomy was performed via laparotomy. After lethal exsanguination of the animals, the pancreas was isolated and frozen. The islet isolation was performed using collagenase / neutral protease (950Wun, respectively). Sch units and 63 units (Serva, Heidelberg, Germany) The digested pancreas was then separated into four discontinuous Euroficoll gradients (Mediat Tech, Manassas, VA) and Cobe 2991 blood cell processor (Carid The final islet preparation samples were counted and purified at 100 bp. The islets were expressed as islet equivalents (IEQ). The isolated islets were cultured overnight, counted, and transplanted in transplant medium ( The mixture was suspended in PBS (Mediatech).

[0224] Rhesus macaques weighing 3-5 kg ​​were cultured for 4 weeks before transplantation. , streptozotocin (1250 mg / m 2 IV;Zanosar,Teva Pa Diabetes was induced using a steroid drug (Commercial Medicines, Irvine, CA). Diabetes mellitus was diagnosed by intravenous glucose tolerance test (IVGTT) with a bolus of 500 mg / kg Glucose levels were confirmed using dextrose and primate C-peptide measurements. C-peptide was monitored at baseline and 10, 3 and 4 days after dextrose injection. Diabetes mellitus is characterized by elevated blood C-peptide levels without detectable serum C-peptide. This was confirmed by measuring glucose levels. Allogeneic transplantation of pancreatic islets was performed. The mean IEQ was 15,745 (±4,063) through the midline small incision. Infusion was via cannulation of the abdominal and mesenteric veins.

[0225] Blood glucose levels were measured twice daily by ear puncture; NPH (Novolin; Novo No rdisk, Princeton, NJ) and glargine (Lantus; Sanofi-Aventis, Bridgewater, NJ) insulin was administered. Fasting blood glucose (FBG) less than 300 mg / dL before transplantation and after graft rejection After transplantation, IVGTTs were performed periodically to monitor graft function. Transplant recipients were subjected to weekly flow cytometry analysis to determine T cell (CD3 V) activity. 450,CD4 PerCP-Cy5.5,CD8 PerCp;BD Bioscie nce) population and B cell (CD20 PE, BD Bioscience) population After islet transplantation, rejection was diagnosed as a FBG level higher than 130 mg / dL for two consecutive days. The primary endpoint was rejection-free islet graft survival.

[0226] Transplant recipients received 2C10R4 and basiliximab (Simulect, Novartis) (Basel, Switzerland) with sirolimus or basiliximab and sirolimus 2C10R4 (50 mg / kg) was administered on postoperative day (POD) 0. Basiliximab (0.3 mg / kg) was administered intravenously on POD 0 and 7. Sirolimus was administered intravenously to achieve a trough level of 5-15 ng / ml. Basiliximab and sirolimus were administered intramuscularly daily until OD 120. All three animals are historical controls (Badell et al., J. Clin. Invest. 120:4520-312, 2010). Two of these historical contrasts Diabetes was induced in two mice (RQz6 and RIb7) by pancreatectomy and oral sirolimus was administered. was administered.

[0227] Treatment with the above regimen resulted in induction therapy with basiliximab alone and maintenance therapy with sirolimus. This resulted in significantly prolonged islet graft survival (Figure 11A) compared to controls (Figure 11B). The median duration of rejection-free graft survival in animals receiving 2C10R4 was 2 80 days in the control animals compared with 8 days in the control animals (p=0.010, Table 3). Morphological data showed that plasma 2C10R4 levels were below 1 μg / ml by POD 100. Sirolimus was discontinued on POD 120, so the longest (3 Surviving recipients receive immunosuppression for approximately 24 weeks before rejection occurs. No clinically significant infectious complications were observed in animals treated with 2C10R4. This result was consistent with the results of the administration of 2C10 IgG4 isotype. The IgG1 isotype of 2C10 (2C10R1) was used as a basilisk. Two additional animals treated with the combination of mab and sirolimus showed survival of 220 and 162 days. The results showed similar prolonged graft survival between the two groups. Given the positive results in this study, the next step is to administer 2C10 as a maintenance therapy. The objective of this study was to evaluate the effect of graft transplantation on graft survival.

[0228] Table 3 [Table 3]

[0229] Blocking the CD40 / CD154 pathway in conjunction with the CD28 / B7 pathway Blockade of the CD40 / CD154 pathway is useful in combination with other costimulatory blocking agents High-affinity antibodies designed to block the CD28 / B7 costimulatory pathway may prove to be effective. Belatacept, a CTLA4-Ig inhibitor, has been shown to be effective in non-human primate models of kidney and islet transplantation. Efficacy has been demonstrated in Phase II and III clinical trials in patients with kidney transplantation. (Larsen et al., Transplantation 90:1528 -35,2010,Vincenti et al.,Am.J.Transplant .10:535-46,2010, Adams et al., J. Immunol.1 74:542-50,2005, Adams et al., Diabetes 51: 265-70, 2002, Larsen et al., Am.J.Transplan t.5:443-53,2005, Vincenti et al., N.Engl.J Med.358:770-81,2005). In the BENEFIT trial, belatacept Excellent renal function was demonstrated in patients treated with acetaminophen; however, these patients developed had a higher survival rate and more severe grade of biopsy-confirmed acute rejection ( Larsen et al.,Transplantation 90:1528-35 ,2010,Vincenti et al.Am.J.Transplant.10: 535-46, 2010). This high rate of acute rejection and the blockade of CD40 and B7 Synergistic effect (Larsen et al., Nature 381:434-8, 1996 ), we next investigated the efficacy of 2C10 and Veratase in kidney transplantation in non-human primates. The efficacy of combination therapy with acetaminophen will be tested.

[0230] [Example 2] Humanized anti-CD40 antibody The present inventors have isolated a novel humanized Ab against CD40, designated h2C10 (humanized 2C1 We developed and characterized a CD40 antibody (antibody 0) and selected it as a functional full antagonist of CD40. The synthetic epitopes are used to activate or deplete B cells or function as partial agonists. so that the molecule is endowed with unique binding properties that distinguish it from competitor molecules that either The initial mouse-primate chimeric version of the antibody demonstrated relevant in vitro and in vivo results. In vivo preclinical studies, e.g., multiple studies in non-human primates (which allow for the evaluation of transplant rejection) Promising efficacy against gliomas-induced thrombocytopenia and prolonged engraftment in both allografts and xenografts, as well as and has demonstrated a favorable safety profile in non-clinical studies The present inventors have also completed humanization of 2C10 (h2C10), which exhibits excellent characteristics. I did.

[0231] To generate a humanized anti-CD40 antibody, the variable region sequences of the mouse antibody 2C10 were used. We searched the human antibody database. Most of the VH sequences were derived from germline antibodies. VH1-46, VH1-69 and VH1-3 (SEQ ID NO: 30), while V L is mostly germline-derived antibody sequences VK3-11 (SEQ ID NO: 31), VK1 Human VH1-3 and VK3-11 were found to be related to VH1-39 and VK6-21. The relatively high frequency and highly important framework positions in the human repertoire Acceptor framework for CDR grafting due to good conservation in Both variable regions were used to clone the CDRs from the mouse 2C10 antibody. Grafting into a donor-acceptor framework allowed the construction of a 3D model. Six murine VH framework residues that differ from their counterparts likely contact the CDRs. The following were identified: M48, A67, L69, A71, K73 and N76. After the analysis, three humanized VH sequences containing 0, 2, and 6 mouse framework residues were obtained. Columns 2C10_h1, 2C10_h2, and 2C10_h3 were designed (Figure 13a). Five mouse VK framework residues likely contact the CDRs: Q1, R After model design, the nucleotide sequences were identified as 0 and 4, respectively. Two humanized VL sequences, 2C10_l1 and 2C1, containing 10 mouse framework residues 0_l2 was designed (Fig. 13b).

[0232] The parent murine 2C10 antibody was humanized by CDR grafting. The germline frameworks VK3-3 and VK3-11 were selected to be acceptors. Three VH sequences and two VL sequences were designed, and a total of six humanized antibodies were produced. , and tested for human CD40 binding.

[0233] The structure of 2C10-heavy chain-3 (2C10_h3) and 2C10-light chain-2 (2C10_l2) The construct bound CD40 at 0.39 nM, within two-fold of that of mouse 2C10 (0.22 nM). The humanized variable regions were found to produce the best antibodies with the highest binding affinity (Table 2). We then constructed a clinical candidate humanized antibody as IgG4 or stabilized IgG4, and used it in the SwiM The vector was cloned into the R expression system.

[0234] Highly productive stable CHO cell lines were isolated by FACS and analyzed by 3 ELISAs and 1 The strain was screened by fed-batch culture at 0.8 Seven clones were isolated that produced more than g / L of humanized 2C10. 3C9-I6 produced approximately 1.2 g / L under non-optimized conditions.

[0235] Construction of antibody expression vectors The humanized VH sequence was synthesized using the vector pFUS, which contains the constant region of the human IgG2 heavy chain. E-CHIg-hG2a (Invivogen) was cloned into the expression vector LB The humanized VK sequence was synthesized by gene synthesis and contained the constant region of the human κ light chain. The expression vector LB303-304 was constructed by cloning the fragment into an expression vector containing the fragment LB303-304. The main and light chains are driven by the human EF1α promoter for strong, constitutive expression in mammalian cells. In addition, the chimeric 2C10 antibody was produced by using mouse VH and VL. Therefore, the expression vectors LB305 and LB306 were constructed in the same manner. Antibody expression vectors are summarized in Table 4.

[0236] Table 4. Antibody expression vectors [Table 4]

[0237] Each vector in Table 4 contains a heavy or light chain expression cassette under the control of the human EF1a promoter. Vectors LB300-302 and LB305 contain the constant region of the human IgG2 heavy chain. The vectors LB308 and LB309 contain the constant region of the human IgG4 heavy chain. The vectors LB303-304 and LB306 contain the human κ light chain region. It contains a constant region.

[0238] Generation of humanized IgG4 antibodies Humanized to maximize binding activity to further minimize potential effector functions The antibodies (2C10_h3 and 2C10_l2) were treated with human IgG4 or stabilized human IgG4 (S2 The heavy chain variable region 2C10_h3 was first converted to a stabilizing mutation S241P. Before transfection, the vector pFUSE-CHIg-hG4, which contains the constant region of the human IgG4 heavy chain, was used. (Invivogen) (Table 4). S241P) was purified from 293F cells after transient transfection. The concentration of IgG4 antibodies was 25-35 mg / L, which was twice as high as that of IgG2 antibodies. The stabilized IgG4 antibody appeared to have half molecules of The DNA and amino acid sequences of the IgG4 antibody are shown in FIG.

[0239] Cloning of humanized IgG4 (S241P) antibody in the SwiMR expression vector SwiMR expression was developed for easy development of antibody-producing cell lines and fluorescently labeled cell differentiation was performed. A switchable membrane reporter for facilitating isolation of high-producing cells by flow cytometry (FACS) is provided. An IRES-mediated bicistronic expression cassette for membrane-anchored GFP was used. The IRES-GFP cassette was placed downstream of the target gene (GOI). The GFP expression level was compared with the expression level of the GOI. High-producing cells were isolated by FACS and then subjected to Cre recombination. The GFP cassette was removed by treatment with ribosomal enzyme. Humanized 2C10 in a stabilized IgG4 format was purified using S The heavy and light chains were cloned into the wiMR expression system to create vector LB312. It was cloned in two separate expression cassettes under the control of the EF1α promoter. The RES-GFP cassette was placed downstream of the heavy chain sequence and flanked by two LoxP sites. This plasmid contained a puromycin resistance gene for selection in mammalian cells. The β-lactamase gene is carried for bacterial growth.

[0240] Stable selection of CHO cells and isolation of high-producing cells 100 ml of CHOS cells (1 × 10 6 cells / ml, Invitrogen) for 12 0 ug of LB312 (linearized by restriction digestion with Asc I) and 120 ul of Free Transfection with style Max Regent (Invitrogen) The cells were selected with 10-20 μg / ml puromycin for 2 weeks. The GFP expression profile of stable pools was characterized by flow cytometry. The top 1% of cells with the highest GFP signal were pooled into a pool containing 100,000 cells. After 2 weeks of culture, pool 1 was analyzed by flow cytometry for GFP expression. The top 1% of cells with the highest GFP signal were analyzed again by cytometry. The pool was selected as pool number 2 containing 100,000 cells. After 2 days of culture, the pool Route number 2 was injected with 2 μM of recombinant membrane-permeable DNA recombinase Cre (TAT-NLS-C The GFP expression profile was measured after 1 week of culture. Approximately 10% of the cells were analyzed for successful removal of the GFP expression cassette from the chromosome. A complete loss of GFP expression was observed. GFP-negative cells were cultured in 384-well plates. After two weeks, approximately 800 colonies were collected from 10 x 384-well plates. Nee has grown up.

[0241] Further humanized antibodies It is also cloned within the VH1-69 and VL1-39 human germline frameworks. Using two CDR-grafted VH sequences and two CDR-grafted VL sequences, Humanized antibodies were also produced. In this further experiment, the inventors produced two types of heavy chains (HB The heavy and light chain sequences were determined by the following methods: HP+KP was used as a positive control. The antibody was transiently expressed in hCD1 and purified by protein A chromatography. 40 bindings were tested.

[0242] FIG. 14 shows the results of 2C10HP and 2C10HB1 in framework 3 and 2C10 Figure 15 shows the amino acid differences between the HB2 constructs. The sequences of the heavy and light chain variable regions are shown in Table 1. The heavy and light chain variable regions are 2C10HP, ... Includes C10HB1, 2C10HB2, 2C10KP, 2C10KB1 and 2C10KB2 Thus, in certain embodiments, the anti-CD40 antibody is one of the following 2C 10H-K combination: 1.2C10HP+2C10KP 2.2C10HB1+2C10KB1 3.2C10HB1+2C10KB2 4.2C10HB1+2C10KP 5.2C10HB2+2C10KB2 6.2C10HB2+2C10KB1 7.2C10HB2+2C10KP 8.2C10HP+2C10KB1 9.2C10HP+2C10KB2 It may include:

[0243] In vitro binding of CD40 by purified antibodies Humanized and chimeric antibodies were transfected into 100 or 200 ml of 293F cells. The antibody was purified after transfection using a protein A column. The cells were purified from conditioned medium harvested 4 days after treatment.

[0244] Measurement of CD40 binding kinetics CD40 binding kinetics were determined by Forte Bio (contracted to Arag). Purified CD40 was biotinylated and streptavidin was used for measurement. Immobilized on a vidin biosensor.

[0245] Bioproduction for in vivo testing After transfection of CHO cells and selection of stably transfected cells The antibody was purified by protein A column, then buffer exchanged (20 mM citrate sodium, 50 mM NaCl, 5% maltose, pH 6.0), filtered through a 0.2 μm filter. Pool number 1 was cultured for 25 min in CD FortiCHO medium (Invitrogen). For setting up large volume wave bag culture The culture medium was rehydrated three times on days 3, 5, and 7 with 10% CD Efficient The final yield of purified antibody was 1000 mg / ml. The antibody was characterized by SDS-PAGE and SEC-HPLC analysis. The monomeric antibody was 99.4% pure.

[0246] Cell line development Single cell colonies were screened by 3 ELISAs and 1 fed-batch production. Throughout the screening process, cells were incubated with CD FortiCHO All colonies from the 384-well plate were transferred to a 96-well plate and maintained in the medium. 1.2 μl of culture medium from each well was used to coat the plates with anti-human Fc antibody. Antibodies were screened in a ELISA plate containing 1000 ribonucleotides. The top 240 clones were selected as the The cells were expanded in ten 24-well plates. After 5 days of culture, 1.2 μl of culture medium was added. The cells were again screened for antibody levels, and the top 60 clones were cultured in 10 x 6 wells. The cells were expanded in a 6-well plate and cultured in a shaking incubator. The well plates were then cultured by passaging the cells 1:10 into a new set of 6-well plates. The original set of 6-well plate cultures were grown to extinction and then analyzed for antibody levels. The top 24 clones in duplicate 6-well plates were measured by ELISA. The clones were expanded in 30 ml of culture medium in a 125 ml shake flask. The feeding strategy was as follows: on days 3, 5, 7, 9, and 11, the feed was with 7.5% Ex-Cell Advanced CHO Feed 1 (Sigma). The top clone, 3C9-I6, showed a production titer of approximately 1.2 g / L.

[0247] In vitro pharmacology of primate chimeric 2C10 and humanized 2C10 The inventors have demonstrated the following key in vitro pharmacological attributes of their lead candidates: Inhibition of B cell activation induced by CD154-CD40 binding No direct activation of B cells High-affinity antagonists of CD40 (e.g., K d is about 10 -10 M or less, about 10 -10 M~ about 10 -9 M, or as described herein) was identified.

[0248] Novel immunization approaches and extensive in vitro screening approaches allow us to The inventors have developed a non-depleting / non-activating antibody against human CD40 that meets these criteria. We identified an anti-CD40 antibody that is an antagonistic antibody.

[0249] In vitro and in vivo studies have shown that the humanized form retains its superior properties was confirmed.

[0250] As described in the previous section, the 2C10 mAb is a human heavy and light chain framework. The original 2C10 mAb was humanized by CDR grafting into the mAb. The humanized 2C10 construct was affinity-matched to human CD40 by Biacore to retain the antibody. The top three humanized 2C10 antibodies all had the same mAb as the parent 2C10 antibody. b showed a slight decrease in affinity, approximately 2-fold compared to b (Table 5). All of these maintained the exceptionally slow dissociation rate of the parent 2C10 mAb. Among the antibodies, clone 2.189.2, which showed the highest affinity at 390 pM, was selected as the lead antibody. The antibody was selected as a target mAb (h2C10).

[0251] Table 5: CD40 receptor binding kinetics of humanized versions of 2C10 [Table 5]

[0252] Comparing the binding kinetics of humanized 2C10 with competitors, the overall affinity of h2C10 was significantly higher. Yet it is significantly better than competitors with affinities in the nanomolar range. The binding affinity of h2C10 was also compared with that of human CD40 and the non-human primate species used in the preclinical evaluation. As shown in Figure 16, h2C10 showed a significant increase in CD40 activity compared with that of other CD40 derived from the same antibody. It has similar affinity for CD40 in these primate species.

[0253] In vivo characterization of primate chimeric 2C10 and humanized 2C10 In vivo pharmacodynamics, pharmacokinetics, and exploratory safety evaluation of 2C10 was performed in rhesus monkeys. s monkey) to develop the primate chimeric construct of 2C10 and the clinical candidate humanized h2C 10 antibodies. Based on in vitro binding kinetics, humanized lead 2C10 ( After selection of mAb 2.189.2;h2C10), we used h2C10 in The compound will be advanced to PK / PD studies in rhesus monkeys to further characterize it. These studies, which cover a wide range of critical experimental endpoints, The data obtained in this study clearly establish the superior properties of h2C10.

[0254] PD, PK, and safety endpoints were investigated in rhesus monkeys. The study was completed in 2016. Key elements of the study plan, including the primary endpoint and objectives, are presented. 6 (Pharmacodynamics (PD) and pharmacokinetics of 2C10 in rhesus monkeys) The primary outcomes and primary study endpoints are summarized in the Related comparisons include: -Effects on blood B and T lymphocyte counts · Humoral immune response to T cell-dependent antigen keyhole limpet hemocyanin (KLH) The effect CD40 receptor occupancy on blood B cells (PD) Pharmacokinetics (PK) Immunogenicity, as assessed by the formation of anti-drug antibodies (ADAs) Exploratory toxicology including CBC, serum chemistry and complete necropsy Including.

[0255] Table 6. PD, PK and safety of 2C10 in rhesus monkeys Sexuality Test [Table 6]

[0256] The results are summarized in the following sections. h2C10 inhibits CD40 T cells without B cell depletion. For the treatment of conditions in which selective blockade of receptor activation is expected to provide therapeutic benefit. It can be used for

[0257] Effects on T cell-mediated immunity h2C10 blocks T cell-dependent antibody responses (TDAR) in vivo. For in vivo demonstration, monkeys were immunized with KLH 6 hours after administration of h2C10. After that, antibody titers against KLH were measured weekly.

[0258] After administering 10 and 25 mg / kg doses of h2C10 to monkeys, KLH challenge was performed. Both IgG and IgM anti-KLH titers were measured weekly up to 28 days after treatment. 17 is the humanized version of 2C10, and the maximum tested dose was 10 mg / kg in most cases. The results show that complete inhibition of KLH antibody responses was achieved at the dose level. Both answers were suppressed.

[0259] Non-depleting effects on B cells The goal in developing antagonist CD40 antibodies is to deplete targeted CD40+ cells. Because of the limitations involved, we were able to determine the effects on lymphocyte subpopulations, particularly The effect on B cells was analyzed.

[0260] Also, treatment of monkeys with 10 or 25 mg / kg of the humanized form of 2C10 (h2C10) The CD40 target is fully saturated by these concentrations of antibody (receptor occupancy). Despite this (see the section on efficacy), it had no discernible depleting effect on B cells. It was.

[0261] Although saturation of 2C10 binding sites on B cells persisted until the final measurement day (day 28), All monkeys receiving either dose of h2C10 showed normal B lymphocyte subpopulations. The T lymphocyte subsets were maintained (Fig. 18). In this study, injection of 2C10 at a low dose of 10 mg / kg significantly increased the activity of B cells (and possibly CD40, a therapeutic target on monocytes and other antigen-presenting cells, is a key factor in the depletion of unwanted B cells. This indicates that the compound can bind persistently without causing any side effects.

[0262] The fact that 2C10 did not deplete B cells was demonstrated by only 3A8 and Chi220. This suggests a clear advantage over competitors.

[0263] Pharmacodynamics CD40 receptor occupancy Binding and occupancy of CD40 targets by primate chimeric and humanized forms of 2C10 were measured using CD2 Flow cytometry was used to identify available binding sites for 2C10 on CD40 on CD40+ B cells. Fluorescently labeled 2C10 and labeled non-competitive anti-CD40 antibody are used. Blood samples were collected on multiple days from control monkeys and primates, chimeric IgG4, or human Cells were collected from monkeys treated with either 2C10 or 2C10 and analyzed by FACS. The degree of receptor occupancy (%) was calculated directly from the recorded mean fluorescence intensity.

[0264] The humanized 2C10 antibody was administered intravenously at single doses of 10 and 25 mg / kg. B cells Surface CD40 on the thyroid was fully saturated by day 3, and no significant changes were observed after either dose of h2C10. The effect was sustained until the final measurement day (day 28) in all monkeys. Representative data from flow cytometry analysis of blood collected from monkeys 28 days after treatment with is shown in Figure 19.

[0265] These results suggest that a single injection of h2C10 at a low dose of 10 mg / kg significantly increased B cell proliferation. These results demonstrate that the CD40 receptor on the IgG4 receptor can be fully saturated for at least 28 days.

[0266] Pharmacokinetics and anti-drug antibody responses Distinction between the pharmacodynamic effects of 2C10 based on CD40 receptor occupancy and the pharmacokinetics of 2C10 To establish a positive correlation, plasma concentrations of 2C10 were measured at the same time receptor occupancy was measured. The plasma concentrations of 2C10 were measured in blood samples. profile, and most importantly, its persistence in plasma (measured by half-life) This measurement allowed for the assessment of the medication required to maintain an effective therapeutic concentration. Frequency guidance is provided.

[0267] Measured in monkeys treated with either 10 mg / kg or 25 mg / kg humanized 2C10 The mean serum concentrations observed are plotted in Figure 20. The data show that animals were The results showed that monkeys were exposed to C10 for approximately 15 days (9-20 days). This half-life is in the range of that expected for therapeutic antibodies in primates. and within the range of those used in clinical trials that have been administered on relatively infrequent dosing schedules (e.g. This should support further monitoring of the complete data set. The model design allows for robust control of the dose and frequency required to sustain effective antibody concentrations. More accurate estimates will be possible in early clinical trials of h2C10.

[0268] Another evaluation of humanized 2C10 was the possibility of generating antibodies (ADA) against h2C10. This is because biologics of epitopes from one species can be transferred to other species (e.g., humanized mAbs). b) to primates, resulting in rapid elimination of the drug from the plasma. In this test, the time course profile shows that there is no measurable anti-2C10 activity during the test. There was no evidence of antibody production against h2C10, as no animals showed titers. It was.

[0269] Preliminary Safety Assessment The experiments described in this section demonstrate the efficacy of h2C1 against immune mechanisms and off-target effects. This was done to examine the potential for adverse outcomes. For biologic therapy, such evaluations are risk mitigation strategies for assessing safety before human exposure to drugs and for clinical trials It is especially important to develop a model for the immune function or pathology of monkeys. The absence of any adverse or undesirable outcomes is indicative of the overall safety assessment of h2C10. Furthermore, in monkeys treated with 2C10, blood test parameters Administer routine tests for changes in, for example, platelet count and serum chemistry parameters. The results were performed several days later and showed no effect of treatment with chimeric 2C10 or humanized 2C10. Two animals were treated twice with 25 mg / kg of primate chimeric 2C10 and showed no treatment-related disease. The animals were evaluated for gross and microscopic evidence of disease; no treatment-related pathological changes were observed. In addition, to rule out thromboembolic complications, All tissues were examined for fibrin deposition by special stains. Importantly, no traces of CD40 receptors were detected. The dose clearly exceeds the dose required for IND-enabling Relatively high doses, close to the dose levels tested in critical toxicology studies conducted in the US, are This preliminary safety evaluation did not reveal any safety issues with h2C10. This indicates that

[0270] These combined data demonstrate that h2C10 inhibits CD40+ without causing unwanted activation or depletion of target cells and without evidence of off-target toxicity A monoclonal antibody intended for the treatment of patients in whom specific inhibition of CD40 activation is desired without the need for CD40 activation. It has been shown to have desirable pharmacodynamic, pharmacokinetic and safety attributes for the body.

[0271] Although specific embodiments of the present invention have been described and illustrated, such embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. It should not be construed as limiting the invention which is to be construed according to the appended claims. All publications and patent applications mentioned in the specification are incorporated by reference in their entirety for all purposes. and all references thereto are expressly incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be so. The invention as described above is incorporated herein by reference in its entirety for all purposes. Although described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate that In view of the teachings of the present invention, one may It will be readily apparent that certain changes and modifications can be made.

Claims

1. A humanized anti-CD40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 23; A humanized anti-CD40 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 21, and the amino acid sequence of the light chain variable region has at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:

23.

3. The dissociation constant (K Ｄ ) is about 1 × 10 －９ The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof has a molecular weight of less than M.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a) an intact immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab')2; and (e) a disulfide-linked Fv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises at least one constant domain selected from the group consisting of: (a) an IgG constant domain; and (b) an IgA constant domain.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises at least one human constant domain.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof binds to the CD40 extracellular domain.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the CD40 is human or rhesus CD40.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which blocks the activation of B lymphocytes by CD154-expressing Jurkat cells in vitro.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which inhibits the expression of CD23, CD80 or CD86 on B lymphocytes.

11. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A vector comprising the polynucleotide described in claim 11.

13. A cell containing the vector described in claim 12.

14. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the polynucleotide according to claim 11, the vector according to claim 12, or the cell according to claim 13; and a pharmaceutically acceptable carrier.

15. 15. The composition of claim 14 for use in suppressing the immune system, treating or preventing transplant rejection, or prolonging the time to transplant rejection in a subject in need thereof.

16. 16. The composition of claim 15, wherein the subject has undergone an organ transplant or is in need of an organ transplant.

17. 17. The composition of claim 16, wherein the organ is selected from the group consisting of heart, kidney, lung, liver, pancreas, intestine, and thymus or a portion thereof.

18. 16. The composition of claim 15, wherein the subject has undergone a tissue transplant or is in need of a tissue transplant.

19. 19. The composition of claim 18, wherein the tissue is bone, tendon, cornea, skin, heart valve, vein, or bone marrow.

20. 16. The composition of claim 15, formulated for administration prior to transplantation.

21. 16. The composition of claim 15, formulated for continuous administration for at least one month after transplantation.

22. 22. The composition of claim 21, formulated for continued administration for at least six months after transplantation.

23. 15. The composition of claim 14 for use in treating or preventing graft-versus-host disease in a subject in need thereof.

24. 15. The composition of claim 14 for use in the treatment or prophylactic treatment of an autoimmune disorder in a subject in need thereof.

25. 25. The composition of claim 24, wherein the autoimmune disorder is associated with or caused by the presence of autoantibodies.

26. The autoimmune disorder may be systemic lupus erythematosus (SLE), CREST syndrome (calcinosis, Raynaud's syndrome, esophageal motility disorders, sclerodactyly, and telangiectasia), opsoclonus, inflammatory myopathies (e.g., polymyositis, dermatomyositis, and inclusion body myositis), systemic sclerosis, primary biliary cirrhosis, celiac disease (e.g., gluten-sensitive enteropathy), dermatitis herpetiformis, Miller-Fisher syndrome, acute motor axonal neuropathy (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, antiphospholipid syndrome, Wegener's granulomatosis, micromyelitis, and inflammatory bowel disease. inflammatory bowel disease, psoriatic arthritis, psoriatic arthritis, rheumatoid arthritis, chronic polyangiitis, Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmune hepatitis, sclerosing myositis, myasthenia gravis, Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, stiff-person syndrome, limbic encephalitis, Isaacs syndrome, Sydenham's chorea, childhood autoimmune streptococcal neuropsychiatric disorders (PANDAS), encephalitis, type 1 diabetes mellitus, neuromyelitis optica, pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease, psoriatic arthritis, Sjogren's syndrome, lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), lupus), multiple sclerosis, reactive arthritis, polymyositis, dermatomyositis, polyendocrine deficiency, Schmidt's syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, adult-onset diabetes mellitus (e.g., type II diabetes), autoimmune disorders in men and women Sexual infertility, ankylosing spondyloarthritis, ulcerative colitis, Crohn's disease, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, juvenile-onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-open heart syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fancier's disease, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis,Interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reactions, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter's syndrome, eczema, lymphomatoid granulomatosis, Behçet's disease, Kaplan's syndrome, Kawasaki disease, dengue fever, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevata, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuchs' cyclitis, IgA nephropathy, Henoch-Henrik's syndrome 25. The composition of claim 24, wherein the disease is selected from the group consisting of Schonlein purpura, graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Hodgkin's and non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulinemia, Epstein-Barr virus infection, mumps, Evans syndrome, and autoimmune gonadal dysfunction.

27. The composition of claim 15 , wherein the subject is a human.

28. 16. The composition of claim 15, formulated for parenteral, intravenous, subcutaneous, intramuscular, transdermal, oral, topical, intrathecal or topical administration.

29. 16. The composition of claim 15, wherein the use further comprises the administration of an immunosuppressant drug within 6 months of administration of the antibody or antigen-binding fragment thereof.

30. The immunosuppressant is a calcineurin inhibitor, tacrolimus, an mTor inhibitor, fingolimod, myriocin, alemtuzumab, rituximab, an anti-CD4 monoclonal antibody, an anti-LFA1 monoclonal antibody, an anti-LFA3 monoclonal antibody, an anti-CD45 antibody, an anti-CD19 antibody, monabatacept, belatacept, indolyl-ASC; azathioprine, lymphocyte immunoglobulin and anti-thymocyte globulin [horse], mycophenolate mofetil. , mycophenolate sodium, daclizumab, basiliximab, cyclophosphamide, prednisone, prednisolone, leflunomide, FK778, FK779, 15-deoxyspergualin, busulfan, fludarabine, methotrexate, 6-mercaptopurine, 15-deoxyspergualin, LF15-0195, bredinin, brequinar, and muromonab-CD3.

31. 31. The composition of claim 30, wherein the calcineurin inhibitor is cyclosporin A or cyclosporin G.

32. 31. The composition of claim 30, wherein the mTor inhibitor is sirolimus, temsirolimus, zotarolimus, or everolimus.

33. The composition of claim 30, wherein the anti-CD45 antibody is an anti-CD45RB antibody.

34. 31. The composition of claim 30, wherein the immunosuppressant is belatacept.

35. 31. The composition of claim 30, wherein the composition and the immunosuppressant are to be administered within one month of each other.

36. 36. The composition of claim 35, wherein the composition and the immunosuppressant are to be administered within one week of each other.

37. 24. The composition of claim 23, wherein the subject is a human.

38. 24. The composition of claim 23, formulated for parenteral, intravenous, subcutaneous, intramuscular, transdermal, oral, topical, intrathecal or topical administration.

39. 24. The composition of claim 23, wherein the use further comprises the administration of an immunosuppressant drug within six months of administering the composition.

40. The immunosuppressant is a calcineurin inhibitor, tacrolimus, an mTor inhibitor, fingolimod, myriocin, alemtuzumab, rituximab, an anti-CD4 monoclonal antibody, an anti-LFA1 monoclonal antibody, an anti-LFA3 monoclonal antibody, an anti-CD45 antibody, an anti-CD19 antibody, monabatacept, belatacept, indolyl-ASC; azathioprine, lymphocyte immunoglobulin and anti-thymocyte globulin [horse], mycophenolate mofetil. , mycophenolate sodium, daclizumab, basiliximab, cyclophosphamide, prednisone, prednisolone, leflunomide, FK778, FK779, 15-deoxyspergualin, busulfan, fludarabine, methotrexate, 6-mercaptopurine, 15-deoxyspergualin, LF15-0195, bredinin, brequinar, and muromonab-CD3.

41. 41. The composition of claim 40, wherein the calcineurin inhibitor is cyclosporin A or cyclosporin G.

42. 41. The composition of claim 40, wherein the mTor inhibitor is sirolimus, temsirolimus, zotarolimus, or everolimus.

43. The composition of claim 40, wherein the anti-CD45 antibody is an anti-CD45RB antibody.

44. 41. The composition of claim 40, wherein the immunosuppressant is belatacept.

45. 41. The composition of claim 40, wherein the composition and the immunosuppressant are to be administered within one month of each other.

46. 46. ​​The composition of claim 45, wherein the composition and the immunosuppressant are to be administered within one week of each other.

47. 25. The composition of claim 24, wherein the subject is a human.

48. 25. The composition of claim 24, formulated for parenteral, intravenous, subcutaneous, intramuscular, transdermal, oral, topical, intrathecal or topical administration.

49. 25. The composition of claim 24, wherein the use further comprises the administration of an immunosuppressant drug within six months of administering the composition.

50. The immunosuppressant is a calcineurin inhibitor, tacrolimus, an mTor inhibitor, fingolimod, myriocin, alemtuzumab, rituximab, an anti-CD4 monoclonal antibody, an anti-LFA1 monoclonal antibody, an anti-LFA3 monoclonal antibody, an anti-CD45 antibody, an anti-CD19 antibody, monabatacept, belatacept, indolyl-ASC; azathioprine, lymphocyte immunoglobulin and anti-thymocyte globulin [horse], mycophenolate mofetil. , mycophenolate sodium, daclizumab, basiliximab, cyclophosphamide, prednisone, prednisolone, leflunomide, FK778, FK779, 15-deoxyspergualin, busulfan, fludarabine, methotrexate, 6-mercaptopurine, 15-deoxyspergualin, LF15-0195, bredinin, brequinar, and muromonab-CD3.

51. 51. The composition of claim 50, wherein the calcineurin inhibitor is cyclosporin A or cyclosporin G.

52. 51. The composition of claim 50, wherein the mTor inhibitor is sirolimus, temsirolimus, zotarolimus, or everolimus.

53. 51. The composition of claim 50, wherein the anti-CD45 antibody is an anti-CD45RB antibody.

54. 51. The composition of claim 50, wherein the immunosuppressant is belatacept.

55. 51. The composition of claim 50, wherein the composition and the immunosuppressant are to be administered within one month of each other.

56. 56. The composition of claim 55, wherein the composition and the immunosuppressant are to be administered within one week of each other.

57. An isolated polypeptide comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

58. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising: (a) culturing a cell transformed with a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 in a culture medium under conditions in which the polynucleotide is expressed; and (b) recovering the polypeptide from the cells or culture medium. A method comprising:

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